EP1578782A2 - Polytherapie a facteurs costimulants - Google Patents

Polytherapie a facteurs costimulants

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Publication number
EP1578782A2
EP1578782A2 EP03800236A EP03800236A EP1578782A2 EP 1578782 A2 EP1578782 A2 EP 1578782A2 EP 03800236 A EP03800236 A EP 03800236A EP 03800236 A EP03800236 A EP 03800236A EP 1578782 A2 EP1578782 A2 EP 1578782A2
Authority
EP
European Patent Office
Prior art keywords
inhibitor
interval
stnfr
tnf
certain embodiments
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03800236A
Other languages
German (de)
English (en)
Other versions
EP1578782A3 (fr
EP1578782A4 (fr
Inventor
Sanjay Deep Khare
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Amgen Inc
Original Assignee
Amgen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amgen Inc filed Critical Amgen Inc
Publication of EP1578782A3 publication Critical patent/EP1578782A3/fr
Publication of EP1578782A2 publication Critical patent/EP1578782A2/fr
Publication of EP1578782A4 publication Critical patent/EP1578782A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152

Definitions

  • the present invention relates to polypeptides involved in the regulation of inflammation and immune response.
  • the invention also relates to combination therapy using molecules involved in B-cell and T-cell stimulation with cytokine antagonists.
  • Inflammatory autoimmune diseases typically involve a complex dysregulation of the biological system.
  • certain proinflammatory cytokines such as TNF and IL-1 are elevated.
  • immune T cells and B cells may also play a role by producing cytokines, chemokines and autoantibodies.
  • Activation of the immune system to the body's self molecules leads to autoimmunity.
  • Certain inflammatory autoimmune diseases are the outcome of interactions that occur between antigen presenting cells (APC) and T cells, and between T and B cells.
  • Certain inflammatory autoimmune disease are also the result of activation of B cells-in addition to the proinflammatory cytokine cascade.
  • antigenic peptides are presented by major histocompatibility complex (MHC) molecules expressed on antigen presenting cells (APCs) to the T cell receptor expressed on T cells.
  • MHC major histocompatibility complex
  • This recognition event may not fully activate T cells.
  • a CD4 interaction to the MHC molecule and 'a second or co-stimulatory' signal may be required for T cell proliferation, cytokine production and intracellular signaling events.
  • Interaction of CD28 and B7 molecules was identified as a second signal to activate T cells.
  • This pathway of immune co-stimulation has a negative regulator, CTLA4, expressed on activated T cells. Treatment with a soluble receptor recombinant protein of CTLA-4 leads to inactivation of immune T and B cells.
  • a method for treating an IL-1 mediated disease comprising administering a therapeutically effective amount of an IL-1 inhibitor and at least one of a B7 inhibitor and a CD28 inhibitor.
  • a method for treating a TNF- ⁇ mediated disease comprising administering a therapeutically effective amount of a TNF- ⁇ inhibitor and at least one of a B7 inhibitor and a CD28 inhibitor.
  • a method for treating an inflammatory or an autoimmune condition comprising administering a therapeutically effective amount of an IL-1 inhibitor and at least one of a B7 inhibitor and a CD28 inhibitor.
  • a method for treating an inflammatory or an autoimmune condition comprising administering a therapeutically effective amount of a TNF- ⁇ inhibitor and at least one of a B7 inhibitor and a CD28 inhibitor.
  • a method for treating an inflammatory or an autoimmune condition comprising administering a therapeutically effective amount of (i) at least one of an AGP3 inhibitor, a BAFFR inhibitor, and a TACI inhibitor, and (ii) at least one of a B7 inhibitor and a CD28 inhibitor.
  • a method for treating an inflammatory or an autoimmune condition comprising administering a therapeutically effective amount of an IL-1 inhibitor, a therapeutically effective amount of a TNF- ⁇ inhibitor, and at least one of a B7 inhibitor and a CD28 inhibitor.
  • Figure 1 shows mean arthritis scores in collagen-induced arthritis (CIA) animals during and after treatment with an IL-1 inhibitor (KIN2) (SEQ ID NO. 3), murine CTLA4-Fc fusion protein, and a combination thereof.
  • KIN2 IL-1 inhibitor
  • SEQ ID NO. 3 murine CTLA4-Fc fusion protein
  • the arrow indicates that animals were administered by injection 100 ⁇ g of KIN2, 100 ⁇ g murine CTLA4-Fc, or 100 ⁇ g each of KIN2 and murine CTLA4- Fc at day 0, +1 , +2, +4, +6, +8 and +10 post onset of disease.
  • 100 ⁇ g is equivalent to 5 mg of therapeutic per Kg weight of animal (5mg/Kg).
  • PBS Phosphate buffered saline
  • FIG. 2 shows mean arthritis scores in CIA animals during and after treatment with an TNF-alpha inhibitor (PEG sTNFR-1 2.6D), murine CTLA4-Fc fusion protein, and a combination thereof.
  • the arrow indicates that animals were administered by injection of 100 ⁇ g of PEG sTNFR-1 2.6D, 100 ⁇ g murine CTLA4-Fc, or 100 ⁇ g each of PEG sTNFR-1 2.6D and murine CTLA4-Fc at day 0, +1 , +2, +4, +6, +8 and +10 post onset of disease.
  • 100 ⁇ g is equivalent to 5 mg of therapeutic per Kg weight of animal (5mg/Kg).
  • PBS Phosphate buffered saline
  • Fc were used as controls.
  • Figure 3A and 3B show levels of type II collagen (CM) specific antibodies present in serum samples taken ten days after the conclusion of the experiment shown in Figure 2.
  • CM type II collagen
  • Figure 4 shows mean arthritis scores in CIA male
  • mice B10.RIII mice modified with vectors expressing IL-1 ra, s-TNFRI (SEQ ID NO. 4) and/or CTLA-4 SEQ ID NO 2).
  • mice were administered viral vectors encoding IL-1 ra, s-TNFRI, CTLA-4 or ⁇ -galactosidase as a control.
  • mice were administered both IL-1 ra and CTLA-4 vectors, s-TNFRI and CTLA-4 vectors, or IL-1ra and s-TNFRI vectors.
  • Figure 5 shows the percent incidence of arthritis in the same groups of mice as in Figure 4.
  • Figure 6 shows levels of type II collagen (Cll) specific antibodies present in bleeds from the same groups of mice as in Figure 4. Mice were bled several weeks after booster of porcine type II collagen and antibodies were assayed as described in Example 3.
  • Cll type II collagen
  • AGP3 peptibody an AGP3 tandem dimer peptide-Fc fusion
  • AGP3 Pb an AGP3 tandem dimer peptide-Fc fusion
  • 50 ⁇ g of murine CTLA4-Fc or a combination of 100 ⁇ g of AGP3 Pb and 50 ⁇ g of murine CTLA4-Fc.
  • 100 ⁇ g is equivalent to 4 mg of therapeutic per Kg weight of animal (4mg/Kg).
  • 50 ⁇ g is equivalent to 2 mg of therapeutic per Kg weight of animal (2mg/Kg).
  • Fc and PBS were used as controls.
  • Figure 8 shows the percent of mice in the treatment groups in Figure 7 with levels of proteinuria exceeding 300mg/dl.
  • the lupus prone mice were tested for proteinuria every 30 days using Albustix® commercial assay (Bayer AG).
  • Figure 10 shows the changes in levels of IgG specific double stranded DNA in serum collected at 30, 60 and 90 days after initial treatment with either AGP3 Pb (an AGP3 tandem dimer peptide-Fc fusion), murine CTLA4-Fc or the combination thereof.
  • AGP3 Pb an AGP3 tandem dimer peptide-Fc fusion
  • murine CTLA4-Fc murine CTLA4-Fc or the combination thereof.
  • the change of dsDNA antibody level was determined by dividing day 30, 60 and 90 antibody levels by their day 0 pre-treatment antibody levels for all ten mice (mean +/-standard error). Serum was collected on those days and assessed for levels of dsDNA antibody using ELISA techniques as described in Example 4.
  • Figure 11 shows the change in levels of IgM specific double-stranded DNA in serum collected at 30, 60 and 90 days after initial treatment with either AGP3 Pb (an AGP3 tandem dimer peptide-Fc fusion), murine CTLA4-Fc or the combination thereof.
  • AGP3 Pb an AGP3 tandem dimer peptide-Fc fusion
  • murine CTLA4-Fc murine CTLA4-Fc or the combination thereof.
  • the levels of dsDNA and change in dsDNA antibody level was determined as described in Figure 10 except IgM specific dsDNA was detected.
  • Figure 12 shows mean arthritis scores in CIA animals during and after treatment with a TNF-alpha inhibitor (PEG sTNFR-1 2.6D), AGP3 Pb (an AGP3 tandem dimer peptide-Fc fusion), and a combination thereof.
  • the animals were administered by injection of 100 ⁇ g of PEG sTNFR-1 2.6D, 100 ⁇ g AGP3 Pb, or 100 ⁇ g each of PEG sTNFR-1 2.6D and AGP3 Pb at day 0, +1 , +2, +4, +6, +8 and +10 post onset of disease.
  • 100 ⁇ g is equivalent to 5 mg of therapeutic per Kg weight of animal (5mg/Kg).
  • FIG. 13 shows mean arthritis scores in CIA animals during and after treatment with an IL-1 inhibitor (KIN2), AGP3 Pb (an AGP3 tandem dimer peptide-Fc fusion), and a combination thereof.
  • KIN2 an IL-1 inhibitor
  • AGP3 Pb an AGP3 tandem dimer peptide-Fc fusion
  • the animals were administered by injection of 100 ⁇ g of KIN2, 100 ⁇ g AGP3 Pb, or 100 ⁇ g each of KIN2 and AGP3 Pb at day 0, +1 , +2, +4, +6, +8 and +10 post onset of disease.
  • 100 ⁇ g is equivalent to 5 mg of therapeutic per Kg weight of animal (5mg/Kg).
  • Phosphate buffered saline (PBS) and Fc were used as controls.
  • Figure 14 shows mean arthritis scores in CIA animals during and after treatment with KIN2, PEG sTNFR-1 2.6D, anti-OX40L antibody, a combination of anti-OX40L antibody and KIN2, and a combination of anti-OX40L antibody and PEG sTNFR-1 2.6D.
  • the animals were administered by injection of 100 ⁇ g of KIN2, 100 ⁇ g of PEG sTNFR-1 2.6D, 100 ⁇ g anti-OX40L antibody, 100 ⁇ g each of KIN2 and anti-OX40L antibody, or 100 ⁇ g each of PEG sTNFR-1 2.6D and anti-OX40L antibody at day 0, +1 , +2, +4, +6, +8 and +10 post onset of disease.
  • 100 ⁇ g is equivalent to 5 mg of therapeutic per Kg weight of animal (5mg/Kg).
  • PBS Phosphate buffered saline
  • Rat IgG were used as controls.
  • mice prescreened for greater than 100 mg/dl protein in the urine.
  • the prescreened mice were injected three times per week for 12 weeks with 100 ⁇ g of AGP3 Pb (an AGP3 tandem dimer peptide-Fc fusion), 50 ⁇ g of murine CTLA4-Fc, or a combination of 100 ⁇ g of AGP3 Pb and 50 ⁇ g of murine CTLA4-Fc.
  • 100 ⁇ g is equivalent to 4 mg of therapeutic per Kg weight of animal (4mg/Kg).
  • 50 ⁇ g is equivalent to 2 mg of therapeutic per Kg weight of animal (2mg/Kg).
  • Fc and PBS were used as controls.
  • Figure 16 shows the severity of proteinuria in (NZBxNZW)
  • F1 mice prescreened for greater than 100 mg/dl protein in the urine The prescreened mice were injected three times per week for 12 weeks with 100 ⁇ g of AGP3 Pb (an AGP3 tandem dimer peptide-Fc fusion), 50 ⁇ g of murine CTLA4-Fc, or a combination of 100 ⁇ g of AGP3 Pb and 50 ⁇ g of murine CTLA4-Fc.
  • 100 ⁇ g is equivalent to 4 mg of therapeutic per Kg weight of animal (4mg/Kg).
  • 50 ⁇ g is equivalent to 2 mg of therapeutic per Kg weight of animal (2mg/Kg).
  • Fc and PBS were used as controls.
  • the mice were tested for proteinuria every week using Albustix® commercial assay (Bayer AG).
  • Figure 17 shows percent survival of (NZBxNZW) F1 mice prescreened for greater than 300 mg/dl protein in the urine.
  • the prescreened mice were injected three times per week for twelve weeks with 100 ⁇ g of AGP3 Pb (an AGP3 tandem dimer peptide-Fc fusion), 50 ⁇ g of murine CTLA4-Fc, or a combination of 100 ⁇ g of AGP3 Pb and 50 ⁇ g of murine CTLA4-Fc.
  • 100 ⁇ g is equivalent to 4 mg of therapeutic per Kg weight of animal (4mg/Kg).
  • 50 ⁇ g is equivalent to 2 mg of therapeutic per Kg weight of animal (2mg/Kg).
  • Fc and PBS were used as controls.
  • Figure 18 shows the severity of proteinuria in (NZBxNZW)
  • F1 mice prescreened for greater than 300 mg/dl protein in the urine The prescreened mice were injected three times per week for 36 weeks with 100 ⁇ g of AGP3 Pb (an AGP3 tandem dimer peptide-Fc fusion), 50 ⁇ g of murine CTLA4-Fc, or a combination of 100 ⁇ g of AGP3.Pb and 50 ⁇ g of murine CTLA4-Fc.
  • 100 ⁇ g is equivalent to 4 mg of therapeutic per Kg weight of animal (4mg/Kg).
  • 50 ⁇ g is equivalent to 2 mg of therapeutic per Kg weight of animal (2mg/Kg).
  • Fc and PBS were used as controls.
  • the mice were tested for proteinuria every week using Albustix® commercial assay (Bayer AG).
  • Figure 19 shows the AGP-3 Peptibody amino acid sequence (SEQ ID NO. 1).
  • Figure 20 shows human CTLA4 amino acid sequence
  • Figure 21 shows the KIN2 (FclL-1 ra) nucleotide (SEQ ID NO:
  • Figure 22 shows sTNFR-l nucleotide (SEQ ID NO. 6) and corresponding amino acid sequence (SEQ ID NO. 4).
  • Figure 23 shows the IL-1 Receptor amino acid sequence
  • Figure 24 shows the TNFR-I amino acid sequence (SEQ ID NO. 7).
  • Figure 25 shows the TNFR-II amino acid sequence (SEQ ID NO:
  • Figure 26 shows the CD40 amino acid sequence (SEQ ID NO:
  • Figure 27 shows the CD30 amino acid sequence (SEQ ID NO:
  • Figure 28 shows the ICOS amino acid sequence (SEQ ID NO:
  • Figure 29 shows the CD28 amino acid sequence (SEQ ID NO:
  • Figure 30 shows the OX40 amino acid sequence (SEQ ID NO:
  • Figure 31 shows the 4-1 -BB amino acid sequence (SEQ ID NO:
  • Figure 32 shows the CD27 amino acid sequence ⁇ SEQ ID NO: 1
  • Figure 33 shows the IL-18 Receptor amino acid sequence (SEQ ID NO. 17).
  • Figure 34 shows the PD-1 amino acid sequence (SEQ ID NO: 1]
  • Figure 35 shows the rat TNFR-I amino acid sequence
  • Figure 36 shows the murine CTLA4 amino acid sequence
  • Figure 37 shows the TACI amino acid sequence (SEQ ID NO:
  • Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.
  • isolated polynucleotide shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotide in which the "isolated polynucleotide” is found in nature, (2) is linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence.
  • isolated protein means a protein encoded by cDNA, recombinant RNA, or synthetic origin or some combination thereof, which (1) is free of at least some proteins with which it would normally be found, (2) is essentially free of other proteins from the same source, e.g., from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature.
  • polypeptide is used herein as a generic term to refer to native proteins, or sequences that have deletions, additions, and/or substitutions of one or more amino acids of the native sequence.
  • naturally-occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from'a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.
  • control sequence refers to polynucleotide sequences which may effect the expression and processing of coding sequences to which they are ligated. The nature of such control sequences may differ depending upon the host organism.
  • control sequences for prokaryotes may include promoter, ribosomal binding site, and transcription termination sequence.
  • control sequences for eukaryotes may include promoters and transcription termination sequence.
  • control sequences can include leader sequences and/or fusion partner sequences.
  • soluble receptor molecule refers to a molecule comprising a fragment of a receptor that remains capable of binding to one or more ligands associated with the receptor.
  • a fragment may comprise the entire extracellular domain of a receptor or a subfragment thereof.
  • the soluble receptor molecules may be linked to other groups.
  • the fragment may be linked to additional amino acids from the receptor, as in splice variants.
  • splice variants may comprise amino acids from the intracellular domain or transmembrane domain, or even from another natural protein.
  • the fragment may be linked to another protein or protein fragment sequence, forming a fusion protein.
  • the fusion partner protein or fragment may provide greater half-life to the molecule (e.g., an Fc domain, albumin, or a leucine zipper domain).
  • the fusion partner protein or fragment may also provide a different functionality (e.g., capable of binding to the same or a different ligand), forming a bifunctional molecule.
  • this functional fusion partner may be another fragment of the same receptor, thus forming a ligand binding dimer.
  • the fragment may be linked to an Fc domain, albumin, or a leucine zipper domain.
  • this functional fusion partner may be another fragment of the same receptor, thus forming a ligand binding dimer.
  • N-terminal methionine which may be useful to allow expression in prokaryotic cells such as E. coli.
  • the fragment may be linked to non-proteinaceous groups.
  • groups include, but are not limited to, N-linked or O-linked carbohydrate chains, water-soluble polymers such as polyethylene glycol (PEG) and derivatives thereof (see for example U.S. Patent No. 4,179,337).
  • PEG polyethylene glycol
  • Other chemical modifications within the meaning of this term include, but are not limited to, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and related molecules.
  • the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
  • polypeptides may also be modified at predetermined positions in the polypeptide, such as at the amino terminus, or at a selected lysine or arginine residue within the polypeptide.
  • Other chemical modifications include, but are not limited to, a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
  • protein refers to polypeptides regardless of length or origin, comprising molecules that are recombinantly produced or naturally occurring, full length or truncated, having a natural sequence or mutated sequence, with or without post-translational n odification, whether produced in mammalian cells, bacterial cells, or any other expression system.
  • binding partner refers to any molecule that preferentially binds to a protein of interest, regardless of the antagonistic or agonistic activity of the molecule toward the protein of interest.
  • specific binding partners include, but are not limited to, antibodies, solubilized receptors, peptides, modified peptides, and related molecules.
  • vehicles refers to a molecule that prevents degradation and/or increases half-life, reduces toxicity, reduces immunogenicity, or increases biological activity of a therapeutic protein.
  • vehicles include an Fc domain, as well as a linear polymer (e.g., polyethylene glycol (PEG), polylysine, dextran, etc.); a branched-chain polymer (see, for example, U.S. Patent No.
  • native Fc refers to a molecule or sequence comprising the sequence of a non-antigen-binding fragment resulting from digestion of whole antibody, whether in monomeric or multimeric form.
  • the original immunoglobulin source of the native Fc is of human origin and may be any of the immunoglobulins, including, but not limited to, lgG1 and lgG2.
  • Native Fc's typically are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association.
  • the number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., lgG1 , lgG2, lgG3, lgA1 , lgGA2).
  • a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see, e.g., Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9).
  • the term "native Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms.
  • Fc variant refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn.
  • International applications WO 97/34631 (published 25 September 1997) and WO 96/32478 describe exemplary Fc variants, as well as interaction with the salvage receptor, and are hereby incorporated by reference in their entirety for any purpose.
  • the term "Fc variant” comprises a molecule or sequence that is humanized from a non- human native Fc.
  • a native Fc comprises sites that may be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention.
  • the term "Fc variant” comprises a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1 ) disulfide bond formation, (2) incompatibility with a selected host cell (3) N- terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).
  • DCC antibody-dependent cellular cytotoxicity
  • Fc domain encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc's, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.
  • multimer as applied to Fc domains or molecules comprising Fc domains refers to molecules having two or more polypeptide chains associated covalently, noncovalently, or by both covalent and non-covalent interactions.
  • IgG molecules typically form dimers; IgM, pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, or tetramers.
  • multimers may be formed by exploiting the sequence and resulting activity of the native Ig source of the Fc/or by derivatizing (as defined below) such a native Fc.
  • the term "dimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two polypeptide chains associated covalently or non-covalently.
  • the terms "derivatizing” and “derivative” or “derivatized” comprise processes and resulting compounds respectively in which at least one of the following is present: (1) the compound has a cyclic portion; for example, cross-linking between cysteinyl residues within the compound; (2) the compound is cross-linked or has a cross-linking site; for example, the compound has a cysteinyl residue and thus forms cross-linked dimers in culture or in vivo; (3) one or more peptidyl linkage is replaced by a non- peptidyl linkage; (4) the N-terminus is replaced by -NRR 1 , NRC(0)R 1 , - NRC(0)OR ⁇ -NRS(0) 2 R ⁇ -NHC(0)NHR, a succinimide group, or substituted or unsubstituted benzyloxycarbonyl-NH-, wherein R and R 1 and the ring substituents are as defined hereinafter; (5) the C-terminus is replaced by - C(0)R 2
  • peptide refers to molecules of 2 to 40 amino acids, including, but not limited to, molecules of 3 to 20 amino acids and molecules of 6 to 15 amino acids.
  • peptides may be randomly generated by any of the methods cited herein, carried in a peptide library (e.g., a phage display library), or derived by digestion of proteins.
  • randomized refers to fully random sequences (e.g., selected by phage display methods) and sequences in which one or more residues of a naturally occurring molecule is replaced by an amino acid residue not appearing in that position in the naturally occurring molecule.
  • Exemplary methods for identifying peptide sequences include, but are not limited to, phage display, E. co// display, ribosome display, yeast-based screening, RNA-peptide screening, chemical screening, rational design, protein structural analysis, and the like.
  • Certain randomized peptides and certain methods of generating them appear in, e.g., WO 00/24782, published May 4, 2000, which is hereby incorporated by reference in its entirety for any purpose.
  • pharmacologically active means that a substance so described is determined to have activity that affects a medical parameter (e.g., T cell proliferation) or disease state (e.g., cancer, autoimmune disorders).
  • pharmacologically active compounds comprise agonistic or mimetic and antagonistic compounds as defined below.
  • pharmaceutical agent or drug refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
  • biological sample includes, but is not limited to, any quantity of a substance from a living thing or formerly living thing.
  • living things include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals.
  • substances include, but are not limited to, blood, serum, urine, cells, organs, tissues, bone, bone marrow, lymph nodes, and skin.
  • substantially pure means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition).
  • a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.
  • a substantially pure composition will comprise more than about 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition.
  • the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists 'essentially of a single macromolecular species. /
  • patient includes human and animal subjects.
  • agent is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.
  • label refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotin moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods).
  • marked avidin e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods.
  • the label or marker can also be therapeutic.
  • Various methods of labeling polypeptides and glycoproteins are known in the art and may be used.
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, ⁇ - galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • antagonist refers to a molecule that blocks or in some way interferes with the biological activity of the associated protein of interest, or has biological activity comparable to a known antagonist or inhibitor of the associated protein of interest.
  • physiologically acceptable salts of the compounds of this invention are also encompassed herein.
  • physiologically acceptable salts is meant any salts that are known or later discovered to be pharmaceutically acceptable. Certain examples include, but are not limited to, acetate; trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide; sulfate; citrate; tartrate; glycolate; and oxalate.
  • polynucleotide as referred to herein means a polymeric form of nucleotides of at least 10 bases in length.
  • the bases may be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide.
  • the term includes single and double stranded forms of DNA.
  • oligonucleotide as referred to herein includes naturally occurring, and modified nucleotides linked together by naturally occurring, and/or non-naturally occurring oligonucleotide linkages. Oligonucleotides are a polynucleotide subset generally comprising a length of 200 bases or fewer.
  • oligonucleotides are 10 to 60 bases in length. In certain embodiments, oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides may be single stranded or double stranded, e.g. for use in the construction of a gene mutant. Oligonucleotides of the invention may be sense or antisense oligonucleotides.
  • nucleotides includes deoxyribonucleotides and ribonucleotides.
  • modified nucleotides includes nucleotides with modified or substituted sugar groups and the like.
  • oligonucleotide linkages includes oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See, e.g., LaPlanche et al. Nucl. Acids Res. 14:9081 (1986); Stec et al.
  • oligonucleotide can include a label for detection.
  • Certain preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are described in publicly available computer programs. Certain computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res., 12:387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wl, BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. BioL, 215:403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, MD 20894; Altschul et al., supra (1990)). The well- known Smith Waterman algorithm may also be used to determine identity.
  • NCBI National Center for Biotechnology Information
  • Certain alignment schemes for aligning two amino acid sequences may result in the matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full- length sequences. Accordingly, in certain embodiments, the selected alignment method (GAP program) will result in an alignment that spans at least 50 contiguous amino acids of the target polypeptide.
  • a gap opening penalty (which is calculated as 3X the average diagonal; the "average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm.
  • a standard comparison matrix (see Dayhoff et al., Atlas of Protein Sequence and Structure, 5(3)(1978) for the PAM 250 comparison matrix; Henikoff etal., Proc. Natl. Acad. Sci USA, 89:10915-10919 (1992) for the BLOSUM 62 comparison matrix) is also used by the algorithm.
  • the parameters for a polypeptide sequence comparison include the following:
  • the GAP program may be useful with the above parameters.
  • the aforementioned parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps) using the GAP algorithm.
  • Examples of unconventional amino acids include: 4-hydroxyproline, ⁇ - carboxyglutamate, ⁇ -N,N,N-trimethyllysine, ⁇ -N-acetyllysine, O- phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, ⁇ -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).
  • the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
  • the left-hand end of single-stranded polynucleotide sequences is the 5' end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction.
  • the direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".
  • modulator is a compound that changes or alters the activity or function of a molecule.
  • a modulator may cause an increase or decrease in the magnitude of a certain activity or function of a molecule compared to the magnitude of the activity or function observed in the absence of the modulator.
  • a modulator is an inhibitor, which decreases the magnitude of at least one activity or function of a molecule.
  • Certain exemplary activities and functions of a molecule include, but are not limited to, binding affinity, enzymatic activity, and signal transduction.
  • Certain exemplary inhibitors include, but are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic molecules. Peptibodies are described, e.g., in WO01/83525.
  • Any number of molecules may serve as specific binding partners within the present invention.
  • Exemplary molecules include, but are not limited to, antibodies, peptides, and Fc-peptide fusion molecules.
  • Antibody or "antibody peptide(s)” refer to an intact antibody, or a binding fragment thereof that competes with the intact antibody for specific binding. In certain embodiments, binding fragments are produced by recombinant DNA techniques. In certain embodiments, binding fragments are produced by enzymatic or chemical cleavage of intact antibodies. Binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, and single- chain antibodies.
  • the invention provides for an antibody or antigen binding domain thereof, or a fragment, variant, or derivative thereof, which binds to an epitope on any of the target molecules and has partial or complete antagonist activity.
  • the target molecule is mammalian, such as human, and may be in soluble or cell surface associated forms, or fragments, derivatives and variants thereof.
  • an antibody may be prepared by immunizing an animal with the target molecule (e.g., murine or human BCMA or TACI) or with an immunogenic fragment, derivative or variant thereof.
  • an animal may be immunized with cells transfected with a vector containing a nucleic acid molecule encoding the target molecule such that the target . molecule is expressed and associated with the surface of the transfected cells.
  • specific binding partners that are antibodies may be obtained by screening a library comprising antibody or antigen binding domain sequences for binding to the target molecule.
  • a library may be prepared in bacteriophage as protein or peptide fusions to a bacteriophage coat protein which are expressed on the surface of assembled phage particles and the encoding DNA sequences contained within the phage particles (so-called "phage display library").
  • phage display library contains DNA sequences encoding human antibodies, such as variable light and heavy chains.
  • sequences binding to the target molecule may be further evolved by multiple rounds of mutagenesis and screening.
  • specific binding partners that are antibodies or antigen binding domains may be tetrameric glycoproteins similar to native antibodies, or they may be single chain antibodies; for example, Fv, Fab, Fab' or F(ab)' fragments, bispecific antibodies, heteroantibodies, or other fragments, variants, or derivatives thereof, which are capable of binding the target molecule and partially or completely neutralizing the target molecule activity.
  • antibodies or antigen binding domains may be produced in hybridoma cell lines (antibody-producing cells such as spleen cells fused to mouse myeloma cells, for example) or may be produced in heterologous cell lines transfected with nucleic acid molecules encoding said antibody or antigen binding domain.
  • Exemplary antibodies include, but are not limited to, polyclonal monospecific polyclonal, monoclonal, recombinant, chimeric, humanized, fully human, single chain and/or bispecific antibodies.
  • antibody fragments include those portions of an antibody that bind to an epitope on a target molecule. Examples of such fragments include, but are not limited to, Fab F(ab'), F(ab)', Fv, and sFv fragments.
  • antibodies may be generated by enzymatic cleavage of full- length antibodies or by recombinant DNA techniques, such as expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.
  • an antigen is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen.
  • An antigen can have one or more epitope.
  • a specific reaction involving an antigen means that an antigen will react, in a selective manner, with its corresponding antibody and not with a multitude of other antibodies which can be evoked by other antigens.
  • polyclonal antibodies directed toward a target molecule generally are raised in animals (e.g., rabbits or mice) by multiple subcutaneous or intraperitoneal injections of the target molecule and an adjuvant.
  • the target molecule, or a variant, fragment, or derivative thereof is conjugated to a carrier protein that is immunogenic in the species to be immunized, such as, in certain embodiments, keyhole limpet heocyanin, serum, albumin, bovine thyroglobulin, or soybean trypsin inhibitor.
  • aggregating agents such as alum may be used to enhance the immune response.
  • the animals may be bled and the serum assayed for anti-target antibody titer.
  • Monoclonal antibodies contain a substantially homogeneous population of antibodies containing substantially similar epitope binding sites. Such antibodies may be of any immunoglobulin class, including, but not limited to, IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • a hybridoma producing a monoclonal antibody of the present invention may be cultivated in vitro, in situ, or in vivo. In certain embodiments, one produces high titers in vivo or in situ.
  • Monoclonal antibodies directed toward the target molecule are typically produced using any method which provides for the production of antibody molecules by continuous cell lines in culture.
  • Exemplary methods for preparing monoclonal antibodies include, but are not limited to, the hybridoma methods of Kohler et al., Nature 256, 495-497 (1975), and the human B-cell hybridoma method, Kozbor, J. Immunol. 133, 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51 -63 (Marcel Dekker, Inc., New York, 1987); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988); the contents of which are incorporated herein' by reference in their entirety for any purpose.
  • specific binding partners include monoclonal antibodies which will inhibit partially or completely the binding of the human target molecule to its cognate ligand or receptor or an antibody having substantially the same specific binding characteristics, as well as fragments and regions thereof.
  • Certain methods for determining monoclonal antibody specificity and affinity by competitive inhibition can be found, e.g., in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. EnzymoL, 92:589-601 (1983). Each of these references is incorporated herein by reference in its entirety for any purpose.
  • hybridoma cell lines produce monoclonal antibodies reactive with target polypeptides.
  • Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
  • chimeric antibodies may be used to reduce immunogenicity in application and to increase yields in production.
  • murine monoclonal antibodies have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric monoclonal antibodies may be used.
  • a chimeric monoclonal antibody may be used as a therapeutic agent.
  • a portion of the heavy and/or light chain may be identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to one particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci., 81, 6851-6855 (1985).
  • chimeric antibody includes monovalent, divalent or polyvalent immunoglobulins.
  • a monovalent chimeric antibody is a dimer (HL) formed by a chimeric H chain associated through disulfide bridges with a chimeric L chain.
  • a divalent chimeric antibody is a tetramer (H 2 L 2 ) formed by two HL dimers associated through at least one disulfide bridge.
  • a polyvalent chimeric antibody may be produced, for example, by employing a CH region that aggregates (e.g., from an IgM H chain, or ⁇ chain).
  • murine and chimeric antibodies, fragments and regions may comprise individual heavy (H) and/or light (L) immunoglobulin chains.
  • a chimeric H chain comprises an antigen binding region derived from the H chain of a non-human antibody specific for the target molecule, which is linked to at least a portion of a human H chain C region (CH), such as CHi or CH 2 .
  • CH human H chain C region
  • a chimeric L chain comprises an antigen binding region derived from the L chain of a non-human antibody specific for the target molecule, linked to at least a portion of a human L chain C region (CL).
  • specific binding partners such as antibodies, fragments, or derivatives, having chimeric H chains and L chains of the same or different variable region binding specificity, can also be prepared by appropriate association of the individual polypeptide chains, according to known method steps, e ⁇ g., according to Ausubel et al., eds. Current Protocols in Molecular Biology, Wiley Interscience, N.Y. (1993), and Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). The contents of these references are incorporated herein by reference in their entirety for any purpose.
  • hosts expressing chimeric H chains (or their derivatives) are separately cultured from hosts expressing chimeric L chains (or their derivatives), and the immunoglobulin chains are separately recovered and then associated.
  • the hosts can be co- cultured and the chains allowed to associate spontaneously in the culture medium, followed by recovery of the assembled immunoglobulin, fragment or derivative.
  • the antigen binding region of the specific binding partner (such as a chimeric antibody) of the present invention may be derived from a non-human antibody specific for the human analog of the target molecule.
  • the sources for the DNA encoding a non-human antibody include cell lines which produce antibodies, such as hybrid cell lines commonly known as hybridomas.
  • the invention also provides for fragments, variants and derivatives, and fusions of anti-target antibodies, wherein the terms “fragments”, “variants”, “derivatives” and “fusions” are defined herein.
  • the invention encompasses fragments, variants, derivatives, and fusions of anti-target antibodies which are functionally similar to the unmodified antibody, that is, they retain at least one of the activities of the unmodified antibody.
  • modifications include the addition of genetic sequences coding for cytotoxic proteins such as plant and bacterial toxins.
  • the fragments, variants, derivatives and fusions of the antibodies can be produced from any host.
  • fragment refers to a peptide or polypeptide that comprises less than the full length amino acid sequence of a protein. Such a fragment may arise, for example, from a truncation at the amino terminus, a truncation at the carboxy terminus, and/or an internal deletion of a residue(s) from the amino acid sequence. Fragments may result from alternative RNA splicing or from in vivo protease activity.
  • variant refers to a peptide or polypeptide comprising one or more amino acid sequence substitutions, deletions, and/or additions as compared to a native or unmodified sequence. Variants may be naturally occurring, such as allelic or splice variants, or may be artificially constructed. Polypeptide variants may be prepared from the corresponding nucleic acid molecules encoding said variants.
  • derivative refers to a polypeptide or peptide, or a variant or fragment thereof, which has been chemically modified. Examples include, but are not limited to, covalent attachment of one or more polymers, such as water soluble polymers, N-linked, or O-linked carbohydrates, sugars, phosphates, and/or other such molecules. In certain embodiments, derivatives are modified in a manner that is different from naturally occurring or starting peptide or polypeptides, either in the type or location of the molecules attached. Exemplary derivatives include, but are not limited to, deletion of one or more chemical groups which are naturally present on the peptide or polypeptide.
  • fusion refers to the joining of a peptide or polypeptide, or fragment, variant and/or derivative thereof, with a heterologous peptide or polypeptide.
  • Suitable fragments include, but are not limited to, for example, Fab, Fab', F(ab') 2 , Fv and scFv. These fragments lack the Fc fragment of an intact antibody, clear more rapidly from the circulation, and can have less non-specific tissue binding than an intact antibody. See Wahl et al., J. Nucl. Med., 24:316-325 (1983).
  • fragments are produced from intact antibodies using methods well known in the art.
  • fragments are produced from intact antibodies by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
  • variants of specific binding partners are also provided.
  • variants of antibodies and antigen binding domains comprise changes in light and/or heavy chain amino acid sequences that are naturally occurring or are introduced by in vitro. engineering of native sequences using recombinant DNA techniques.
  • naturally occurring variants include "somatic" variants which are generated in vivo in the corresponding germ line nucleotide sequences during the generation of an antibody response to a foreign antigen.
  • variants of antibodies and antigen binding domains may be prepared by mutagenesis techniques known in the art.
  • amino acid changes may be introduced at random throughout an antibody coding region and the resulting variants may be screened for a desired activity, such as binding affinity for the target molecule.
  • amino acid changes may be introduced in selected regions of an antibody, such as in the light and/or heavy chain CDRs, and framework regions, and the resulting antibodies may be screened for binding to the target molecule or some other activity.
  • Amino acid changes encompass one or more amino acid substitutions in a CDR, ranging from a single amino acid difference to the introduction of all possible permutations of amino acids within a given CDR, such as CDR3.
  • each residue within a CDR to target binding may be assessed by substituting at least one residue within the CDR with alanine (Lewis et al. (1995), Mol. Immunol. 32: 1065-72).
  • residues which are not optimal for binding to the target molecule may then be changed in order to determine a more optimum sequence.
  • variants may be generated by insertion of amino acids to increase the size of a CDR, such as CDR3.
  • most light chain CDR3 sequences are nine amino acids in length.
  • light chain CDR3 sequences in an antibody which are shorter than nine residues may be optimized for binding to the target molecule by insertion of appropriate amino acids to increase the length of the CDR.
  • antibody or antigen binding domain variants comprise one or more amino acid changes in one or more of the heavy or light chain CDR1 , CDR2 or CDR3 and optionally one or more of the heavy or light chain framework regions FR1 , FR2 or FR3.
  • amino acid changes comprise substitutions, deletions and/or insertions of amino acid residues.
  • variants may be prepared by
  • chain shuffling of either light or heavy chains. Marks et al. (1992), Biotechnology .0: 779-83.
  • a single light (or heavy) chain is combined with a library having a repertoire of heavy (or light) chains and the resulting population is screened for a desired activity, such as binding to the target molecule.
  • This technique may permit screening of a greater sample of different heavy (or light) chains in combination with a single light (or heavy) chain than is possible with libraries comprising repertoires of both heavy and light chains.
  • the specific binding partners of the invention may be bispecific.
  • Bispecific specific binding partners can be of several configurations.
  • bispecific antibodies resemble single antibodies (or antibody fragments) but have two different antigen binding sites (variable regions).
  • bispecific antibodies can be produced by chemical techniques (See e.g., Kranz et al., Proc. Natl. Acad. Sci. USA, 78:5807 (1981)), by "polydoma” techniques (see U.S. Pat. No. 4,474,893 to Reading) or by recombinant DNA techniques.
  • the specific binding partners of the invention may also be heteroantibodies.
  • Heteroantibodies are two or more antibodies, or antibody binding fragments (Fab) linked together, each antibody or fragment having a different specificity.
  • humanized antibodies are provided. Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into a human antibody from a source which is non- human. In general, non-human residues will be present in CDRs.
  • humanization can be performed following methods known in the art (See, e.g., Jones et al., Nature 321, 522-525 (1986); Riechmann et al., Nature, 332, 323-327 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988)), by substituting rodent complementarily-determining regions (CDRs) for the corresponding regions of a human antibody.
  • CDRs rodent complementarily-determining regions
  • humanization of the mouse humoral immune system.
  • Introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated offers the opportunity to study the mechanisms underlying programmed expression and assembly of antibodies as well as their role in B- cell development.
  • Ig immunoglobulin
  • MAbs monoclonal antibodies
  • fully human antibodies are expected to minimize the immunogenic and allergic responses intrinsic to mouse or mouse-derivatized Mabs, and thus, in certain embodiments, increase the efficacy and safety of the administered antibodies.
  • fully human antibodies may be used in the treatment of chronic and recurring human diseases, such as osteoporosis, inflammation, autoimmunity, and cancer, which may involve repeated antibody administrations.
  • chronic and recurring human diseases such as osteoporosis, inflammation, autoimmunity, and cancer, which may involve repeated antibody administrations.
  • the reproduced human antibody repertoire in these mouse strains may yield high affinity antibodies against any antigen of interest, including human antigens.
  • antigen-specific human MAbs with the desired specificity may be produced and selected.
  • specific binding partners including chimeric, CDR-grafted, and humanized antibodies
  • nucleic acids encoding the antibodies are introduced into host cells and expressed using materials and procedures described herein and known in the art.
  • the antibodies are produced in mammalian host cells, such as CHO cells.
  • fully human antibodies may be produced by expression of recombinant DNA transfected into host cells or by expression in hybridoma cells as described above.
  • antibody-specific . messenger RNA molecules are extracted from immune system cells taken from an immunized animal, and transcribed into complementary DNA (cDNA).
  • cDNA complementary DNA
  • the cDNA is then cloned into a bacterial expression system.
  • a bacteriophage lambda vector system having a leader sequence that causes the expressed Fab protein to migrate to the periplasmic space (between the bacterial cell membrane and the cell wall) or to be secreted may be used.
  • target molecule specific binding partners Fab fragments with specificity for the target molecule
  • antibody as it is defined, discussed, and claimed herein.
  • chimeric antibodies may be produced by splicing the genes from a mouse antibody molecule of appropriate antigen-specificity together with genes from a human antibody molecule of appropriate biological activity, such as the ability to activate human complement and mediate ADCC.
  • a Fc region may be replaced with that of a different isotype. Specific binding partners such as antibodies produced by this technique are within the scope of certain embodiments of the invention.
  • the antibodies are fully human antibodies.
  • the antibodies that bind target molecules are encoded by nucleic acid sequences which are naturally occurring somatic variants of human germline immunoglobulin nucleic acid sequence, and fragments, synthetic variants, derivatives and fusions thereof. Such antibodies may be produced by any method known in the art.
  • antibodies may be produced by immunization with a target antigen (any target polypeptide capable of eliciting an immune response, and optionally conjugated to a carrier) of transgenic animals (e.g., mice) that are capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production. See, for example, Jakobovits et al., Proc.
  • human antibodies may be generated through the in w ⁇ r ⁇ screening of phage display antibody libraries. See Hoogenboom et al., J. Mol. BioL, 227, 381 (1991 ); Marks et al., J. Mol. BioL, 222, 581 (1991), incorporated herein by reference.
  • libraries may contain a diversity of human antibody sequences, such as human Fab, Fv, and scFv fragments, that may be screened against an appropriate target.
  • phage display libraries may comprise peptides or proteins other than antibodies which may be screened to identify specific binding partners of the target molecule.
  • An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
  • An Id antibody may be prepared by immunizing an animal of the same species and genetic type (e.g., mouse strain) as the source of the monoclonal antibody with the monoclonal antibody to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody). See, for example, U.S. Pat. No. 4,699,880, which is herein incorporated by reference in its entirety for any purpose.
  • the anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-ld antibody.
  • the anti- anti-ld may be epitopically identical to the original monoclonal antibody which induced the anti-Id.
  • Naturally occurring antibody stru tural units typically comprise a tetramer.
  • Each such tetramer typically is /composed of two identical pairs of polypeptide chains, each pair having one full-length "light” (in certain embodiments, about 25 kDa) and one full-length "heavy” chain (in certain embodiments, about 50-70 kDa).
  • the amino-terminal portion of each chain typically includes a variable region of about 100 to 110 or more amino acids that typically is responsible for antigen recognition.
  • the carboxy- terminal portion of each chain typically defines a constant region that may be responsible for effector function.
  • Human light chains are typically classified as kappa and lambda light chains.
  • Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • IgG has several subclasses, including, but not limited to, lgG1 , lgG2, lgG3, and lgG4.
  • IgM has subclasses including, but not limited to, lgM1 and lgM2.
  • IgA is similarly subdivided into subclasses including, but not limited to, lgA1 and lgA2.
  • variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids.
  • J Fundamental Immunology Ch. 7
  • D variable region of about 10 more amino acids.
  • variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by three hyper variable regions, also called complementarity determining regions or CDRs.
  • the CDRs from the two chains of each pair typically are aligned by the framework regions, which may enable binding to a specific epitope.
  • both light and heavy chain variable regions typically comprise the domains FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4.
  • the assignment of amino acids to each domain is typically in accordance with the definitions of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).
  • the term “heavy chain” includes any polypeptide having sufficient variable region sequence to confer specificity for an antigen.
  • the term “light chain” includes any polypeptide having sufficient variable region sequence to confer specificity for an antigen.
  • a full-length heavy chain includes a variable region domain, VH, and three constant region domains, CH1 , CH2, and CH3. The VH domain is at the amino-terminus of the polypeptide, and the CH3 domain is at the carboxy-terminus.
  • the term “heavy chain”, as used herein, encompasses a full-length heavy chain and fragments thereof.
  • a full-length light chain includes a variable region domain, VL, and a constant region domain, CL.
  • variable region domain of the light chain is at the amino-terminus of the polypeptide.
  • a Fab fragment is comprised of one light chain and the CH1 and variable regions of one heavy chain.
  • the heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • a Fab' fragment contains one light chain and one heavy chain that contains more of the constant region, between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between two heavy chains to form a F(ab')2 molecule.
  • the Fv region comprises the variable regions from both the heavy and light chains, but lacks the constant regions.
  • Single-chain antibodies are Fv molecules in which the heavy and light chain variable regions have been connected by a flexible linker to form a single polypeptide chain which forms an antigen-binding region.
  • Single chain antibodies are discussed in detail in WO 88/01649 and U.S. Patent Nos. 4,946,778 and 5,260,203.
  • multifunctional antibody in certain embodiments, typically is understood to have each of its binding sites identical.
  • An antibody substantially inhibits adhesion of a ligand to a receptor when an excess of antibody reduces the quantity of receptor bound to counterreceptor by at least about 20%, 40%, 60%, k ⁇ %, 85%, or more (as measured in an in vitro competitive binding assay).
  • epitope includes any polypeptide determinant capable of specific binding to an immunoglobulin or T-cell receptor.
  • epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics.
  • An epitope is a region of an antigen that is bound by an antibody.
  • an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
  • an antibody is said to specifically bind an antigen when the dissociation constant is ⁇ 1 ⁇ M, in certain embodiments, when the dissociation constant is ⁇ 100 nM, and in certain embodiments, when the dissociation constant is ⁇ 10 nM.
  • a peptide used as a specific binding partner may be comprised within a molecule of the formula
  • F 1 is a vehicle
  • X 1 and X 2 are each independently selected from -(L 1 ) c -P 1 , -(L 1 ) c - P 1 -(L 2 ) d -P 2 , -(L 1 ) c -P 1 -(L 2 ) d -P 2 -(L 3 )e-P 3 , and -(L 1 ) c -P 1 -(L 2 ) d -P 2 -(L 3 ) e -P 3 -(L 4 ) f -P 4
  • P 1 , P 2 , P 3 , and P 4 are each independently peptide sequences, wherein at least one is a specific binding partner;
  • L 1 , L 2 , L 3 , and L 4 are each independently linkers; and a, b, c, d, e, and f are each independently 0 or 1 , provided that at least one of a and b is 1.
  • a molecule comprises a structure of the formula
  • a molecule comprises a structure of the formula
  • the vehicle is an Fc domain.
  • the Fc domain may be IgG Fc.
  • the IgG Fc domain may be lgG1.
  • soluble receptor fragments Another class of specific binding partners are soluble receptor fragments. Certain soluble receptor fragments are identified in the figures: a. the IL-1 receptor (SEQ ID NO. 7). b. TNFRI (SEQ ID NO. 8). c. TNFRII (SEQ ID NO. 9). d. CD40 (SEQ ID NO. 10). e. CD30 (SEQ ID NO. 11). f. ICOS (SEQ ID NO. 12). g. CD28 (SEQ ID NO. 13). h. OX40 (SEQ ID NO. 14). i. 4-1-BB (SEQ ID NO. 15). j. CD27 (SEQ ID NO. 16). k. the IL-18 receptor (SEQ ID NO. 17). '
  • these specific binding partners may be covalently linked to a vehicle. In certain embodiments, these specific binding partners may be covalently linked to an Fc domain.
  • peptide and polypeptide sequences may be from conservative and/or non-conservative modifications of the amino acid sequences of certain native molecules.
  • conservative modifications may produce molecules having functional and chemical characteristics similar to those of the molecule from which such modifications are made.
  • substantial modifications in the functional and/or chemical characteristics of the molecules may be accomplished by selecting substitutions in the amino acid sequence that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the molecule.
  • a "conservative amino acid substitution” may involve a substitution of a native amino acid residue with a nonnative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.
  • any native residue in the polypeptide may also be substituted with alanine, as has been previously described for "alanine scanning mutagenesis” (see, for example, MacLennan et al., 1998, Ada Physiol. Scand. Suppl. 643:55-67; Sasaki et al., 1998, Adv. Biophys. 35:1-24, which discuss alanine scanning mutagenesis).
  • Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.
  • amino acid substitutions can be determined by those skilled in the art at the time such substitutions are desired.
  • amino acid substitutions can be used to identify important residues of the molecule sequence, or to increase or decrease the affinity of the molecules described herein.
  • Naturally occurring residues may be divided into classes based on common side chain properties:
  • non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.
  • Such substituted residues may be introduced into regions of the human antibody that are homologous with non-human antibodies, or into the non-homologous regions of the molecule.
  • the hydropathic index of amino acids may be considered.
  • Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as in the present case.
  • the greatest local average hydrophilicity of a protein as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.
  • hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (- 1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4).
  • the substitution of amino acids whose hydrophilicity values are within ⁇ 2 is included, in certain embodiments, those which are within ⁇ 1 are included, and in certain embodiments, those within ⁇ 0.5 are included.
  • a skilled artisan will be able to determine suitable variants of the polypeptide as set forth herein using well-known techniques.
  • one skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity.
  • even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
  • One skilled in the art can also analyze the three- dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of an antibody with respect to its three dimensional structure. In certain embodiments, one skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants could be used to gather information about suitable variants.
  • One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity of greater than 30%, or similarity greater than 40% often have similar structural topologies.
  • the recent growth of the protein structural database (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure. See Holm et al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been suggested (Brenner et al., Curr. Op. Struct. BioL, 7(3):369-376 (1997)) that there are a limited number of folds in a given polypeptide or protein and that once a critical number of structures have been resolved, structural prediction will become dramatically more accurate.
  • Additional methods of predicting secondary structure include “threading” (Jones, D., Curr. Opin. Struct. BioL, 7(3):377-87 (1997); Sippl et al., Structure, 4(1):15-19 (1996)), “profile analysis” (Bowie et al., Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13) .4355-4358 (1987)), and “evolutionary linkage” (See Holm, supra (1999), and Brenner, supra (1997)).
  • antibody variants include glycosylation variants wherein the number and/or type of glycosylation site has been altered compared to the amino acid sequenbes of the parent polypeptide.
  • protein variants comprise a greater or a lesser number of N-linked glycosylation sites than the native protien.
  • An N- linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn- X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline.
  • the substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions which eliminate this sequence will remove an existing N-linked carbohydrate chain.
  • N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created.
  • Additional preferred antibody variants include cysteine variants wherein one or more cysteine residues are deleted from or substituted for another amino acid (e.g., serine) as compared to the parent amino acid sequence. Cysteine variants may be useful when antibodies must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.
  • amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and/or (4) confer or modify other physiocochemical or functional properties on such polypeptides.
  • single or multiple amino acid substitutions may be made in the naturally-occurring sequence (in certain embodiments, in the portion of the polypeptide outside the domain(s) forming intermolecular contacts).
  • a conservative amino acid substitution typically may not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).
  • a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence.
  • Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991 )); and Thornton et at. Nature 354:105 (1991), which are each incorporated herein by reference.
  • polypeptide fragment refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion. In certain embodiments, fragments are at least 5 to 467 amino acids long. It will be appreciated that in certain embodiments, fragments are at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 150, 200, 250, 300, 350, 400, or 450 amino acids long.
  • Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics" or "peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger TINS p.392 (1985); and Evans et al. J. Med. Chem. 30:1229 (1987), which are incorporated herein by reference for any purpose. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce a similar therapeutic or prophylactic effect.
  • a paradigm polypeptide i.e., a polypeptide that has a biochemical property or pharmacological activity
  • Systematic substitution of one or more amino acids of a consensus sequence with a D- amino acid of the same type may be used in certain embodiments to generate more stable peptides.
  • constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo and Gierasch Ann. Rev. Biochem. 61 :387 (1992), incorporated herein by reference for any purpose); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
  • a nucleic acid molecule encoding an antibody or antigen binding domain is inserted into an appropriate expression vector using standard ligation techniques.
  • the vector may be selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene can occur).
  • a nucleic acid molecule encoding an antibody may be amplified/expressed in prokaryotic, yeast, insect (baculovirus systems) and/or eukaryotic host cells.
  • selection of the host cell will take into account, in part, whether an antibody is to be post-translationally modified (e.g., glycosylated and/or phosphorylated).
  • yeast, insect, or mammalian host cells are selected to facilitate post- translational modifications.
  • expression vectors see, e.g., Meth. Enz. v. 185, (D.V. Goeddel, ed.), Academic Press Inc., San Diego, CA (1990).
  • expression vectors used in any host cells will contain one or more of the following components: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.
  • a promoter one or more enhancer sequences
  • an origin of replication a transcriptional termination sequence
  • a complete intron sequence containing a donor and acceptor splice site a leader sequence for secretion
  • a ribosome binding site a polyadenylation sequence
  • polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed
  • Exemplary vector components include, but are not limited to, homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of different sequences from more than one source), synthetic, or native sequences which normally function to regulate immunoglobulin expression.
  • a source of vector components may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the components are functional in, and can be activated by, the host cell machinery.
  • an origin of replication is selected based upon the type of host cell being used for expression.
  • the origin of replication from the plasmid pBR322 (Product No. 303-3s, New England Biolabs, Beverly, MA) is suitable for most Gram-negative bacteria while, in certain embodiments, various origins from SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV) or papillomaviruses (such as HPV or BPV) are useful for cloning vectors in mammalian cells.
  • the origin of replication component is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it contains the early promoter).
  • a transcription termination sequence may be located 3' of the end of a polypeptide coding regions and serves to terminate transcription.
  • a transcription termination sequence in prokaryotic cells is a G-C rich fragment followed by a poly T sequence. While certain sequences may be easily cloned from a library or even purchased commercially as part of a vector, in certain embodiments, sequences can also be readily synthesized using methods for nucleic acid synthesis such as those described above.
  • a selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium.
  • selection marker genes include those that encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells, (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media.
  • Exemplary selectable markers include, but are not limited to, the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene.
  • a neomycin resistance gene may be used for selection in prokaryotic and eukaryotic host cells.
  • selectable markers for mammalian cells may be dihydrofolate reductase (DHFR) and thymidine kinase.
  • DHFR dihydrofolate reductase
  • thymidine kinase thymidine kinase
  • Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of selection partner in the medium is successively changed, thereby leading to amplification of both the selection gene and the DNA that encodes an antibody.
  • increased quantities of an antibody are synthesized from the amplified DNA.
  • a ribosome binding site is typically present for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes).
  • the element is typically located 3' to the promoter and 5' to the coding sequence of the polypeptide to be expressed.
  • the Shine-Dalgarno sequence is varied but is typically a polypurine (i.e., having a high A-G content). Many Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using methods set forth above and used in a prokaryotic vector.
  • a leader or signal sequence may be used to direct secretion of a polypeptide.
  • a signal sequence may be positioned within or directly at the 5' end of a polypeptide coding region. Many signal sequences have been identified and, in certain embodiments, may be selected based upon the host cell used for expression.
  • a signal sequence may be homologous (naturally occurring) or heterologous to a nucleic acid sequence encoding an antibody or antigen binding domain.
  • a heterologous signal sequence selected should be one that is recognized and processed, i.e., cleaved by a signal peptidase, by the host cell.
  • the signal sequence may be substituted with a prokaryotic signal sequence selected, e.g., from alkaline phosphatase, penicillinase, or heat-stable enterotoxin II leaders.
  • a native immunoglobulin signal sequence may be substituted by a yeast leader sequence.
  • Exemplary yeast leader sequences include, but are not limited to, yeast invertase, alpha factor, or acid phosphatase leaders.
  • mammalian cell expression using the native signal sequence may be satisfactory.
  • other mammalian signal sequences may be suitable.
  • secretion of an antibody or antigen binding domain from a host cell will result in the removal of the signal peptide from the antibody.
  • the mature antibody will lack any leader or signal sequence.
  • the final protein product may have, in the -1 position (relative to the first amino acid of the mature protein) one or more additional amino acids incident to expression, which may not have been totally removed.
  • the final protein product may have one or two amino acids found in the peptidase cleavage site, attached to the N-terminus.
  • use of some enzyme cleavage sites may result in a slightly truncated form of the desired polypeptide, if the enzyme cuts at such area within the mature polypeptide.
  • the expression vectors may contain a promoter that is recognized by the host organism and operably linked to a nucleic acid molecule encoding an antibody or antigen binding domain.
  • a native or heterologous promoter may be used depending on the host cell used for expression and the yield of protein desired.
  • Exemplary promoters for use with prokaryotic hosts include, but are not limited to, beta-lactamase and lactose promoter systems; alkaline phosphatase; a tryptophan (trp) promoter system; and hybrid promoters such as the tac promoter.
  • other known bacterial promoters may be used. The sequences of known bacterial promoters have been published, thereby enabling one skilled in the art to ligate them to the desired DNA sequence(s), using linkers or adapters as needed to supply any desired restriction sites.
  • Suitable promoters for use with yeast hosts are also well known in the art.
  • yeast enhancers are advantageously used with yeast promoters.
  • Suitable promoters for use with mammalian host cells are well known.
  • Exemplary promoters for use with mammalian host cells include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40).
  • Exemplary mammalian promoters include, but are not limited to, heterologous mammalian promoters.
  • Exemplary heterologous mammalian promoters include, but are not limited to, heat-shock promoters and the actin promoter.
  • Exemplary promoters which may be used for expressing specific binding partners include, but are not limited to, the SV40 early promoter region (Benoist and Chambon (1981), Nature, 290:304-310); the CMV promoter; the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al. (1980), Cell, 22: 787-97); the herpes thymidine kinase promoter (Wagner et al. (1981), Proc. Natl. Acad. Sci. U.S.A., 78: 1444-5); the regulatory sequences of the metallothionine gene (Brinster et al.
  • prokaryotic expression vectors such as the beta-lactamase promoter (Villa-Kamaroff et al. (1978), Proc. Natl. Acad. Sci. U.S.A., 75: 3727-31); and the tac promoter (DeBoer, et al. (1983), Proc. Natl. Acad. Sci. U.S.A., 80: 21-25).
  • animal transcriptional control regions which exhibit tissue specificity may be used in transgenic animals.
  • Exemplary transcriptional control regions for use with tissue specific expression in transgenic animals include, but are not limited to, the elastase I gene control region which is active in pancreatic acinar cells (Swift et al. (1984), Cell, 38: 639-46; Ornitz et al. (1986), Cold Spring Harbor Symp. Quant. Biol.
  • albumin gene control region which is active in liver (Pinkert et al. (1987), Genes and DeveL, 1 : 268-76); the alphafetoprotein gene control region which is active in liver (Krumlauf et al. (1987), Mol. Cell. Bio ⁇ ., 5: 1639-48; Hammer et al. (1987), Science, 235: 53-58); the alpha 1 -antitrypsin gene control region which is active in the liver (Kelsey et al. (1987), Genes and DeveL, 1 : 161- 171 ); the beta-globin gene control region which is active in myeloid cells (Mogram et al.
  • an enhancer sequence may be inserted into the vector to increase transcription in eucaryotic host cells.
  • Exemplary enhancer sequences from mammalian genes include, but are not limited to, globin, elastase, albumin, alpha-feto-protein, and insulin.
  • an enhancer from a virus will be used.
  • Exemplary enhancer sequences for the activation of eukaryotic promoters include, but are not limited to, the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers are exemplary enhancing elements.
  • an enhancer may be spliced into the vector at a position 5' or 3' to the polypeptide coding region. In certain embodiments, the enhancer is located at a site 5' from the promoter.
  • vectors are those which are compatible with at least one of bacterial, insect, and mammalian host cells.
  • Exemplary vectors include, but are not limited to, pCRII, pCR3, and pcDNA3.1 (Invitrogen Company, San Diego, CA), pBSII (Stratagene Company, La Jolla, CA), pET15 (Novagen, Madison, Wl), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBacll; Invitrogen), pDSR-alpha (PCT Publication No. WO90/14363) and pFastBacDual (Gibco/BRL, Grand Island, NY).
  • Exemplary vectors include, but are not limited to, cosmids, plasmids and modified viruses compatible with the selected host cell.
  • the vectors may include plasmids including, but not limited to, Bluescript® plasmid derivatives (a high copy number ColE1 - based phagemid, Stratagene Cloning Systems Inc., La Jolla CA), PCR cloning plasmids designed for cloning Taq-amplified PCR products (e.g.,
  • the recombinant molecules may be introduced into host cells via transformation, transfection, infection, electroporation, or other known techniques.
  • host cells may be prokaryotic host cells (such as E. coli) or eukaryotic host cells (such as a yeast cell, an insect cell, or a vertebrate cell).
  • prokaryotic host cells such as E. coli produce unglycosylated protein; for example, unglyclosylated shBCMA and unglycosylated shTACI, which may possess advantages over the glycosylated eukaryotic molecules.
  • the host cell when cultured under appropriate conditions, expresses an antibody or antigen binding domain of the invention which can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted).
  • selection of an appropriate host cell will take into account various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity, such as glycosylation or phosphorylation, and/or ease of folding into a biologically active molecule.
  • a number of suitable host cells are known in the art and many are available from the American Type Culture Collection (ATCC), Manassas, VA.
  • exemplary host cells include, but are not limited to, mammalian cells, such as Chinese hamster ovary cells (CHO) (ATCC No. CCL61) CHO DHFR- cells (Urlaub et al. (1980), Proc) Natl. Acad. Sci. USA 97, 4216-20), human embryonic kidney (HEK) 293 or;293T cells (ATCC No. CRL1573), and 3T3 cells (ATCC No. CCL92).
  • CHO Chinese hamster ovary cells
  • CHO DHFR- cells Urlaub et al. (1980), Proc) Natl. Acad. Sci. USA 97, 4216-20
  • HEK human embryonic kidney
  • ATCC No. CRL1573 ATCC No. CCL92
  • Exemplary host cells include, but are not limited to, the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), and the CV-1 cell line (ATCC No. CCL70).
  • Exemplary mammalian host cells include, but are not limited to, primate cell lines and rodent cell lines, including transformed cell lines.
  • Exemplary host cells include, but are not limited to, normal diploid cells, cell strains derived from in vitro culture of primary tissue, and primary explants.
  • candidate cells may be genotypically deficient in the selection gene, or may contain a dominantly acting selection gene.
  • Exemplary host cells include, but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines, which are available from the American Type Culture Collection, Manassas, VA). Each of these cell lines is known by and available to those skilled in the art of protein expression.
  • the host cells may be bacterial cells.
  • Exemplary bacterial host cells include, but are no limited to, various strains of E. coli (e.g., HB101 , (ATCC No. 33694) DH5 , DH10, and MC1061 (ATCC No. 53338)).
  • Exemplary host cells also include, but are not limited to, various strains of Pseudomonas spp., B. subtilis, other Bacillus spp., Streptomyces spp.
  • yeast cells Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of polypeptides.
  • the host cell may be Saccharomyces cerivisae.
  • insect cell systems may be used.
  • insect cells include, but are not limited to, Sf-9 and Hi5 (Invitrogen, Carlsbad, CA).
  • transformation or transfection of a nucleic acid molecule encoding a specific binding partner into a selected host cell may be accomplished by well known methods including methods such as calcium chloride, electroporation, microinjection, lipofection or the DEAE-dextran method.
  • the method selected will in part be a function of the type of host cell to be used.
  • transgenic animals may be used to express glycosylated specific binding partners, such as antibodies and antigen binding domain.
  • glycosylated specific binding partners such as antibodies and antigen binding domain.
  • one may use a transgenic milk-producing animal (a cow or goat, for example) and obtain glycosylated binding partners in the animal milk.
  • one may use plants to produce glycosylated specific binding partners.
  • Host cells comprising (as by transformation or transfection) an expression vector encoding a specific binding partner of the target molecule may be cultured using standard media well known to the skilled artisan.
  • the media may contain all nutrients necessary for the growth and survival of the cells.
  • E. coli cells may be cultured in Luria Broth (LB) and/or Terrific Broth (TB).
  • Exemplary media for culturing eukaryotic cells include, but are not limited to, RPMI 1640, MEM, DMEM, all of which may be supplemented with serum and/or growth factors according to the particular cell line being cultured.
  • insect cells may be cultured in Grace's medium supplemented with yeastolate, lactalbumin hydrolysate, and/or fetal calf serum.
  • an antibiotic or other compound useful for selective growth of transfected or transformed cells is added as a supplement to the media.
  • the compound to be used is chosen in view of the selectable marker element present on the plasmid with which the host cell was transformed.
  • the selectable marker element is kanamycin resistance
  • the compound added to the culture medium will be kanamycin.
  • Exemplary compounds for selective growth include, but are not limited to, ampicillin, tetracycline and neomycin.
  • the amount of an antibody or antigen binding domain produced by a host cell can be evaluated using standard methods known in the art. Exemplary methods include, but are not limited to, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, HPLC separation, immunoprecipitation, and activity assays.
  • purification of a specific binding partner that has been secreted into the cell media may be accomplished using a variety of techniques including affinity, immunoaffinity or ion exchange chromatography, molecular sieve chromatography, preparative gel electrophoresis or isoelectric focusing, chromatofocusing, and high pressure liquid chromatography.
  • antibodies comprising a Fc region may be conveniently purified by affinity chromatography with Protein A, which selectively binds the Fc region.
  • modified forms of an antibody or antigen binding domain may be prepared with affinity tags, such as hexahistidine or other small peptide such as FLAG (Eastman Kodak Co., New Haven, CT) or myc (Invitrogen) at either the carboxyl or amino terminus and purified by a one-step affinity column.
  • affinity tags such as hexahistidine or other small peptide such as FLAG (Eastman Kodak Co., New Haven, CT) or myc (Invitrogen) at either the carboxyl or amino terminus and purified by a one-step affinity column.
  • polyhistidine binds with great affinity and specificity to nickel, thus an affinity column of nickel (such as the Qiagen® nickel columns) can be used for purification of polyhistidine-tagged specific binding partners. See for example, Ausubel et al., eds. (1993), Current Protocols in Molecular Biology, Section 10.11.8, John Wiley & Sons, New York. In certain embodiments, more than
  • specific binding partners which are expressed in procaryotic host cells may be present in soluble form either in the periplasmic space or in the cytoplasm or in an insoluble form as part of intracellular inclusion bodies.
  • specific binding partners can be extracted from the host cell using any standard technique known to the skilled artisan.
  • the host cells can be lysed to release the contents of the periplasm/cytoplasm by French press, homogenization, and/or sonication followed by centrifugation.
  • soluble forms of an antibody or antigen binding domain present either in the cytoplasm or released from the periplasmic space may be further purified using methods known in the art.
  • Fab fragments are released from the bacterial periplasmic space by osmotic shock techniques.
  • an antibody or antigen binding domain may often bind to the inner and/or outer cellular membranes and thus will be found primarily in the pellet material after centrifugation.
  • the pellet material may then be treated at pH extremes or with a chaotropic partner such as a detergent, guanidine, guanidine derivatives, urea, or urea derivatives in the presence of a reducing partner such as dithiothreitol at alkaline pH or tris carboxyethyl phosphine at acid pH to release, break apart, and solubilize the inclusion bodies.
  • the soluble specific binding partner may then be analyzed using gel electrophoresis, immunoprecipitation or the like.
  • a solublized antibody or antigen binding domain may be isolated using standard methods such as those set forth below and in Marston et al. (1990), Meth. Enz., 182: 264-75.
  • an antibody or antigen binding domain may not be biologically active upon isolation.
  • methods for "refolding" or converting the polypeptide to its tertiary structure and generating disulfide linkages may be used to restore biological activity.
  • the biological activity may be restored by exposing the solubilized polypeptide to a pH usually above 7 in the presence of a particular concentration of a chaotrope.
  • the selection of chaotrope is very similar to the choices used for inclusion body solubilization, but, in certain embodiments, the chaotrope is used at a lower concentration and is not necessarily the same as chaotropes used for the solubilization.
  • the refolding/oxidation solution will also contain a reducing partner or the reducing partner plus its oxidized form in a specific ratio to generate a particular redox potential allowing for disulfide shuffling to occur in the formation of the protein's cysteine bridge(s).
  • exemplary redox couples include, but are not limited to, cysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride, dithiothreitol(DTT)/dithiane DTT, and 2- mercaptoethanol(bME)/dithio-b(ME).
  • a cosolvent may be used or may be needed to increase the efficiency of the refolding and exemplary repartners used for this purpose include, but are not limited to, glycerol, polyethylene glycol of various molecular weights, arginine, and related molecules.
  • specific binding partners may be prepared by chemical synthesis methods.
  • the chemical synthesis method may incorporate solid phase peptide synthesis.
  • the chemical synthesis methods may use techniques known in the art such as those set forth by Merrifield et al. (1963), J. Am. Chem. Soc, 85: 2149; Houghten et al. (1985), Proc Natl Acad. Sci. USA, 82: 5132; and Stewart and Young (1984), Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, IL.
  • polypeptides may be synthesized with or without a methionine on the amino terminus.
  • chemically synthesized antibodies and antigen binding domains may be oxidized using methods set forth in these references to form disulfide bridges.
  • antibodies so prepared will retain at least one biological activity associated with a native or recombinantly produced antibody or antigen binding domain.
  • single anti-cytokine therapy or a combination of anti-cytokine therapies have been successfully used, but with some limitations because of activated immune T and B cells. In certain instances, these therapies also involve regular treatment during the course of chronic diseases.
  • Interleukin-1 is an anti-inflammatory cytokine.
  • IL-1 is a mediator in many diseases and medical conditions.
  • IL-1 is manufactured by cells of the macrophage/monocyte lineage.
  • IL-1 is produced in two forms: IL-1 alpha (IL-1 ⁇ ) and IL-1 beta (IL-1 ⁇ ).
  • a disease or medical condition is considered to be an
  • interleukin-1 mediated disease if the spontaneous or experimental disease or medical condition is associated with elevated levels of IL-1 in bodily fluids or tissue and/or if cells or tissues taken from the body produce elevated levels of IL-1 in culture.
  • interleukin-1 mediated diseases are also recognized by the following additional two conditions: (1) pathological findings associated with the disease or medical condition can be mimicked experimentally in animals by administration of IL-1 or upregulation of expression of IL-1 ; and (2) a pathology induced in experimental animal models of the disease or medical condition can be inhibited or abolished by treatment with agents that inhibit the action of IL-1.
  • one or more of the above conditions are met in an IL-1 -mediated disease.
  • all three of the conditions are met in an IL-1 -mediated disease. '
  • Acute and chronic interleukin-1 (IL-1) -mediated diseases include, but are not limited to, the following: acute pancreatitis; amyotrophic lateral sclerosis (ALS, or Lou Gehrig's disease); Alzheimer's disease; cachexia anorexia, including, but not limited to, AIDS-induced cachexia; asthma and other pulmonary diseases; atherosclerosis; autoimmune vasculitis; chronic fatigue syndrome; Clostridium associated illnesses, including, but not limited to, Clostridium-associated diarrhea; coronary conditions and indications, including, but not limited to, congestive heart failure, coronary restenosis, myocardial infarction, myocardial dysfunction (e.g., related to sepsis), and coronary artery bypass graft; cancer, including, but not limited to, leukemias, including, but not limited to, multiple myeloma leukemia and myelogenous (e.g., AML and CML), and tumor metastasis; diabetes (including, but not limited, le
  • an IL-1 inhibitor may be any protein or molecule capable of specifically preventing activation of cellular receptors to IL-1 , which may result from any number of mechanisms.
  • Exemplary mechanisms include, but are not limited to, downregulating IL-1 production, binding free IL-1 , interfering with IL-1 binding to its receptor, interfering with formation of the IL-1 receptor complex (i.e., association of IL-1 receptor with IL-1 receptor accessory protein), and interfering with modulation of IL-1 signaling after binding to its receptor.
  • interleukin-1 inhibitors include, but are not limited to, IL-1 receptor antagonists, including, but not limited to, KineretTM, IL-1 ra, IL- 1 ra variants, and IL-1 ra derivatives, which are collectively termed "IL-1 ra proteins;” anti-IL-1 receptor monoclonal antibodies (see, e.g., EP 623674, which is hereby incorporated by reference for any purpose); IL-1 binding proteins, including, but not limited to, soluble IL-1 receptors (see, e.g., U. S. Pat. No. 5,492,888, U. S. Pat. No. 5,488,032, and U. S. Pat. No. 5,464,937, U.
  • IL-1 receptor antagonists including, but not limited to, KineretTM, IL-1 ra, IL- 1 ra variants, and IL-1 ra derivatives, which are collectively termed "IL-1 ra proteins;” anti-IL-1 receptor
  • anti-IL-1 monoclonal antibodies see, e.g., WO 9501997, WO 9402627, WO 9006371 , U.S.Pat. No.
  • Exemplary IL-1 inhibitors are disclosed, e.g., in US Pat.
  • Interleukin-1 receptor antagonist (IL-1 ra) is a human protein that acts as a natural inhibitor of interleukin-1 and is a member of the IL-1 family, which includes IL-1 ⁇ and IL-1 ⁇ . Certain receptor antagonists, including IL-1 ra and variants and derivatives thereof, as well as methods of making and using them, are described in U.S. Patent No.
  • an IL-1 receptor antagonist may be glycosylated. In certain embodiments, an IL- 1 receptor antagonist may be non-glycosylated.
  • IL-1 ra ⁇ Three forms of IL-1 ra and variants thereof are described in U.S. Pat. No. 5,075,222 (the '222 patent).
  • the first form IL-1 ra ⁇ (called “IL- 1 i" in the '222 patent), is characterized as a 22-23 kD molecule on SDS-PAGE with an approximate isoelectric point of 4.8, which elutes from a Mono Q FPLC column at around 52 mM NaCl in Tris buffer, pH 7.6.
  • the second form, IL-1 ra ⁇ is characterized as a 22-23 kD protein, which elutes from a Mono Q column at 48 mM NaCl.
  • IL-1 ra ⁇ and IL-1 ra ⁇ are glycosylated.
  • the third form, IL-1 rax is characterized as a 20 kD protein, which elutes from a Mono Q column at 48 mM NaCl and is non-glycosylated.
  • the '222 patent also describes certain methods for isolating genes that code for the inhibitors, cloning those genes in suitable vectors, transforming and transfecting those genes into certain cell types, and expressing those genes to produce the inhibitors.
  • KIN2 is a variant of IL-1 ra fused to a Fc molecule.
  • the sequence of KIN2 is disclosed in SEQ ID NO. 3. Further information on expression and purification of KIN2 is in U.S. Patent No. 6,294,170.
  • deletions, insertions, and/or substitutions are made within the amino acid sequences of IL-1 ra.
  • an IL-1 ra variant is biologically active (e.g., possesses the ability to inhibit IL-1).
  • TNF and may be categorized as inflammatory conditions.
  • a "TNF-mediated disease” includes, but is not limited to, a disease or medical condition that is associated with elevated levels of TNF in bodily fluids or tissue and/or in which cells or tissues taken from the body produce elevated levels of TNF in culture.
  • a disease is a TNF- mediated disease if (1) pathological findings associated with the disease or medical condition can be mimicked experimentally in animals by the administration or upregulation of expression of TNF and/or (2) a pathology induced in experimental animal mpdels of the disease or medical condition can be inhibited or abolished by treatment with agents that inhibit the action of TNF.
  • Certain acute and chronic TNF-mediated diseases include, but are not limited to: cachexia and anorexia; cancer, including, but not limited to, leukemia; chronic fatigue syndrome; coronary conditions and/or indications, including, but not limited to, congestive heart failure, coronary restenosis, myocardial infarction, myocardial dysfunction (including but not limited to, such condition related to sepsis), and coronary artery bypass graft; depression; diabetes, including, but not limited to, juvenile onset Type 1 diabetes, diabetes mellitus, and insulin resistance (including, but not limited to, insulin resistance associated with obesity); endometriosis, endometritis, and related conditions; fibromyalgia and analgesia; g ⁇ aft versus host rejection; hyperalgesia; inflammatory bowel diseases, including, but not limited to, Crohn's disease and Clostridium difficile-associated diarrhea; ischemia, including, but not limited to, cerebral ischemia, which includes, but is not limited to,
  • TNF inhibitors may act by at least one of downregulating or inhibiting TNF production, binding free TNF, interfering with TNF binding to its receptor, and interfering with modulation of TNF signaling after binding to its receptor.
  • TNF inhibitor includes, but is not limited to, solubilized TNF receptors, including, but not limited to, soluble tumor necrosis factor receptor type I (sTNFR-1; also called the p55 receptor), soluble tumor necrosis factor receptor type II (also called the p75 receptor), and Enbrel®; antibodies to TNF, including, but not limited to, RemicadeTM and D2E7 (see, e.g., U.S. Patent Nos.
  • TNF- ⁇ inhibitors are described, e,g., in
  • EP 393 438 and EP 422 339 describe the amino acid and nucleic acid sequences of a soluble TNF receptor type I (also known as sTNFR-1 or 30kDa TNF inhibitor) and a soluble TNF receptor type II (also known as sTNFR-ll or 40kDa TNF inhibitor), which are collectively termed "sTNFRs”.
  • sTNFRs soluble TNF receptor type I
  • sTNFR-ll also known as sTNFR-ll or 40kDa TNF inhibitor
  • EP 393 438 and EP 422 339 also describe modified forms of sTNFR-1 and sTNFR-ll, including, but not limited to fragments, functional derivatives, and variants.
  • EP 393 438 and EP 422 339 describe methods for isolating genes that code for the inhibitors, cloning the genes into suitable vectors, transforming or transfecting the genes into certain cell types, and expressing the genes to produce the inhibitors.
  • sTNFR-1 and sTNFR-ll are members of the nerve growth factor/TNF receptor superfamily of receptors, which includes the nerve growth factor receptor (NGF), the B cell antigen CD40, 4-1 BB, the rat T-cell antigen MRC OX40, the fas antigen, and the CD27 and CD30 antigens (Smith et al. (1990) Science, 248:1019-1023).
  • NGF nerve growth factor receptor
  • CD40 the B cell antigen CD40
  • 4-1 BB the rat T-cell antigen MRC OX40
  • the fas antigen the CD27 and CD30 antigens
  • CD27 and CD30 antigens CD27 and CD30 antigens
  • EP 393 438 teaches a 40kDa TNF inhibitor ⁇ 51 and a
  • 40kDa TNF inhibitor ⁇ 53 which are truncated versions of the full-length recombinant 40kDa TNF inhibitor protein.
  • ⁇ 51 and ⁇ 53 have 51 or 53 amino acids, respectively, deleted from the carboxyl terminus of the mature protein.
  • the truncated sTNFRs include the proteins represented by the formula R ⁇ -[Cys 19 -Cys 103 ]-R2 and R4-[Cys 32 - Cys 115 ]-R 5 . These proteins are truncated forms of sTNFR-l and sTNFR-ll, respectively.
  • R r [Cys 19 -Cys 103 ]-R 2 represents one or more proteins wherein [Cys 19 -Cys 103 ] is residues 19 through 103 of sTNFR-l, the sequence of which is provided in Figure 1 of WO 98/01555; wherein Ri represents a methionylated or nonmethionylated amine group of Cys 19 or one or more amino-terminal amino acid residues selected from Cys 18 to Asp 1 ; and wherein R 2 represents a carboxy group of Cys 103 or one or more carboxy- terminal amino acid residues selected from Phe 104 to Leu 110 .
  • Exemplary truncated sTNFR-l's of the present invention include, but are not limited to, sTNFR-l 2.6D/C105, sTNFR-l 2.6D/C106, sTNFR-l 2.6D/N105, sTNFR-l 2.3D/d8, sTNFR-l 2.3D/d18, sTNFR-l 2.3D/d15, either methionylated or nonmethionylated, and variants and derivatives thereof.
  • Certain exemplary truncated sTNFR-1 's are described, e.g., in published PCT Application No. WO 98/01555.
  • R 3 -[Cys 32 -Cys 115 ]-R 4 represents one or more proteins wherein [Cys 32 -Cys 115 ] is residues Cys 32 through Cys 115 of sTNFR-ll, the sequence of which is provided in Figure 8 of WO 98/01555; wherein R 3 represents a methionylated or nonmethionylated amine group of Cys 32 or one or more amino-terminal amino acid residues selected from Cys ⁇ 1 to Leu 1 ; and wherein R4 represents a carboxy group of Cys 115 or one or more carboxy-terminal amino acid residues selected from Ala 116 to Arg 122 .
  • sTNFR-1 may be pegylated.
  • N-terminal pegylated forms of sTNFR-1 are described, e.g., in published PCT Application No. WO 98/01555.
  • Interferons were originally named for their ability to interfere with viral infection of host cells (Isaacs and Lindenman, 1957, Proc. R. Soc. 147:28-267). Since their discovery, a number of members of the interferon family have been identified with various biological roles in addition to antiviral defense, including cell growth and cell immunity. The members of the IFN family were originally classified into three groups from the cell or tissue of origin: leukocyte, fibroblast, and immune. Thereafter, these interferon species became known as IFN- ⁇ , IFN- ⁇ , and IFN- ⁇ , respectively (Pestka et al., 1997, Semin. Oncol. 24(suppl 9):S9-4-S9-17).
  • IFN- ⁇ , IFN- ⁇ , and IFN- ⁇ , and IFN-T bind the type I IFN receptor, while IFN- ⁇ binds the type II IFN receptor (Pfeffer et al., 1998, Cancer Res. 58:2489-2499).
  • Type I IFNs are produced by virtually every cell type and can be induced upon exposure to viruses, double- stranded RNA, polypeptides, and cytokines (Jonasch and Haluska, 2001 , The Oncologist 6:34-55).
  • Type II IFN- ⁇ is primarily produced in T lymphocytes and natural killer (NK) cells and can be induced by a number of immunological stimuli (Id.).
  • IFN- ⁇ signaling depends on at least five distinct proteins:
  • IFNGR1 and IFNGR2 subunits of the IFN- ⁇ receptor
  • Jak1 , Jak2 and the transcription factor STAT1 (Schindler and Darnell, 1995, Annu. Rev. Biochem. 64.621-651 ; Bach et al., 1997, Annu. Rev. Immunol. 15:563-591).
  • IFN- ⁇ receptors are found on most cell types, except mature erythrocytes (Farrar and Schreiber, 1993, Annu. Rev. Immunol. 11:571-611). Jak1 , Jak2, and STAT1 proteins mediate IFN- ⁇ signaling.
  • IFN- ⁇ regulates a variety of biological functions, such as antiviral responses, cell growth, and tumor suppression. Antiviral activity appears to affect all phases of viral infection, including entry, uncoating, transcription, RNA stability, translation, maturation, and assembly and release (Id.). IFN- ⁇ regulates cell growth by inducing or inhibiting apoptosis, depending on the cell type. For example, IFN- ⁇ induces apoptosis of murine pre-B cells but inhibits apoptosis of B chronic lymphocytic leukemia cells (Granch et al., 1993, Eur. J. Immunol.
  • IFN- ⁇ also promotes either cell proliferation or apoptosis in malignant human T cells (Novelli et al., 1994, J. Immunol. 152:496-504).
  • IFN- ⁇ or its effector molecules contribute to pathology seen in human autoimmune disorders.
  • IFN- ⁇ or molecules whose expression is up-regulated by IFN- ⁇ , such as IP-10, HLA DQ, and neopterin are present at increased levels in systemic lupus erythematosus (SLE) patients, particularly those with lupus nephritis (Funauchi et al., 1991 , Tohoku J. Exp. Med. 164:259-267: Yokoyama et al., 1992, Kidney Int. 42:755-763; Al-Janadi et al., 1993, J. Clin.
  • SLE systemic lupus erythematosus
  • mice spontaneously develop disease and a recent report has identified an interferon-inducible gene as a candidate gene involved in predisposition to disease in the NZB strain of mice (Rozzo et al., 2001 , Immunity 15:435-43).
  • IFN- ⁇ modulation/blockade has been performed with a variety of reagents including antibodies to IFN- ⁇ , soluble receptor for IFN- ⁇ (both protein and DNA constructs), and gene knockouts. Positive effects have been repeatedly documented as measured by auto-antibody production, proteinuria, histology, and survival (Theophilopoulos et al., 2001 , Arthritis Res. 3:136-).
  • IFN- ⁇ activity is essential for proper regulation of the immune response.
  • increased IFN- ⁇ activity can result in a pathological condition, such as inflammatory, infectious, and autoimmune disorders and diseases.
  • Inhibition of excessive IFN- ⁇ activity is a promising strategy for treating patients with IFN- ⁇ mediated diseases.
  • IL-18 is a pro-inflammatory cytokine that was found to induce interferon- ⁇ and was previously named interferon gamma inducing factor (IGIF).
  • IGIF interferon gamma inducing factor
  • IL-1 has been shown to upregulate IL-18 production, and IL-18 induces production of a number of proinflammatory cytokines, including IL-6 and MMP-1.
  • caspase I is also important for IL-18 production.
  • TNF- ⁇ regulates IL-18 production, and that simultaneous inhibition of TNF- ⁇ and IL-18 protects against liver toxicity. See, e.g., Faggioni et al. (2000), PNAS 97: 2367-72.
  • IL-18 acts in vivo through a receptor system reminiscent of the IL-1 system.
  • IL-18 interacts with a cell surface receptor (IL-18R), which interacts with an accessory protein (IL-18RAcP).
  • IL-18-mediated signaling proceeds upon formation of the complex of IL-18, IL-18R, and IL-18RAcP.
  • a natural inhibitor for IL-18 is IL-18bp.
  • IL-18bp acts as a "decoy receptor" by binding to IL-18 molecules and preventing interaction with IL-18R.
  • the present invention is directed to therapies comprising at least one IL-18 inhibitor and a B7 inhibitor, a CD28 inhibitor, or both, and methods of treatment using such therapies.
  • a therapy comprises an IL-18 inhibitor and at least one additional molecule described herein.
  • Exemplary conditions that may be treated according to certain embodiments include, but are not limited to, inflammation, autoimmune diseases, IL-1 mediated diseases, and TNF- mediated diseases.
  • Exemplary conditions that may be treated with at least one IL-18 inhibitor and at least one molecule described herein according to certain embodiments include, but are not limited to, arthritis, including, but not limited to rheumatoid arthritis; systemic lupus erythematosus (SLE); graft versus host disease (GvHD); hepatitis; sepsis; and the loss of bone and cartilage accompanying these diseases.
  • Exemplary IL-18 inhibitors include, but are not limited to, antibodies that bind to IL-18; antibodies that bind to IL-18R; antibodies that bind to IL-18RAcP; IL-18bp; IL-18R fragments (e.g., a solubilized extracellular domain of the IL-18 receptor); peptides that bind to IL-18 and reduce or prevent its interaction with IL-18R; peptides that bind to IL-18R and reduce or prevent its interaction with IL-18 or with IL-18RAcP; peptides that bind to IL- 18RAcP and reduce or prevent its interaction with IL-18R; and small molecules that reduce or prevent IL-18 production or the interaction between any of IL-18, IL-18R, and IL-18RAcP.
  • antibodies that bind to IL-18 include, but are not limited to, antibodies that bind to IL-18; antibodies that bind to IL-18R; antibodies that bind to IL-18RAcP; IL-18bp; IL-18R
  • B7 is a receptor that mediates costimulation of T cells.
  • B7 binds to two separate ligands, CD28 and CTLA4. Interaction of B7 and CD28 activates T cells.
  • CTLA4 is a negative regulator of this activation.
  • B7.1 and B7.2 There are at least two B7 receptors, termed B7.1 and B7.2.
  • B7 refers to B7.1 , B7.2, or both B7.1 and B7.2.
  • soluble B7 molecules may be used to modulate costimulatory pathways.
  • soluble CTLA4 molecules may be used to modulate costimulatory pathways.
  • Certain modulators of B cell-T cell costimulatory pathways include, but are not limited to, inhibitors of CD28, B7.1 , and B7.2. Certain examples include, but are not limited to, soluble forms of B7.1 or B7.2 and CTLA4.
  • B7 receptors are also described in WO 92/00092 and WO
  • CD28 sequence is disclosed in Aruffo et al., Proc.
  • CD28 molecules are also disclosed in WO 90/05541 , WO 93/19767, WO 94/28912 and EP 0 445 228.
  • B7.1 and B7.2 are expressed on the surface of activated B lymphocytes (Linsley et al. J Exp. Med. 173, 721-730 (1991); Freeman et al. Science 262, 909-911 (1993))
  • Both B7.1 and B7.2 are receptors for two ligands, CD28 and CTLA4, expressed on T lymphocytes.
  • CD28 is constitutively expressed on resting T cells and increases after activation whereas CTLA4 is not expressed on resting T cells but appears after activation (Brunet et al.
  • CTLA4-lg fusion proteins have been constructed and used to study the effects of CTLA4 binding to B7.1 and B7.2 (see for example WO93/00431 and W097/28267).
  • the B7:CD28/CTLA4 costimulatory pathway has been implicated in T cell mediated immune responses and manipulation of the pathway is useful in the prevention and treatment of a variety of disorders, including rheumatoid arthritis, graft versus host disease, graft rejection, lupus, multiple sclerosis, psoriasis and others, such as IL-1 and TNF-alpha mediated disorders mentioned herein.
  • Immune responses mediated by B cell/T cell costimulatory pathway may be modulated by inhibitors of the CD28/B7 pathway.
  • CTLA4 is fused to a human immunoglobulin region either directly or through one or more linker moieties.
  • CTLA4 comprises an extracellular domain of CTLA4 which binds B7.1 and/or B7.2 and partially or completely inhibits immune responses mediated by the CD28/B7 pathway.
  • a CTLA4 extracellular domain comprises about amino acid residues 1 (methionine), to 124 (aspartic acid) as shown in SEQ ID NO. 2.
  • CTLA4 polypeptides include, but are not limited to, fragments which encompass at least a portion of a CTLA4 extracellular domain, which fragments bind B7.1 and/or B7.2 and partially or completely inhibit immune responses mediated by the CD28/B7 pathway.
  • a CTLA4 extracellular domain may be fused to a human immunoglobulin region either directly or through one or more linker moieties.
  • CTLA4 polypeptides include variants having a substitution, deletion or insertion of one or more amino acids in the sequence shown in SEQ ID NO. 2.
  • a CTLA4 variant may have a substitution of a different amino acid for serine at position 25, alanine at position 29, threonine at position 30, leucine at position 104 and/or glycine at position 105.
  • Certain nonlimiting CTLA4 variants are as described in WO02/02638.
  • a CLTA4 variant has a tyrosine substituted for an alanine at position 29 and a glutamic acid subsituted for a leucine at position 104 of the sequences shown in SEQ ID NO. 2.
  • the abovementioned CTLA4 variants are in the extracellular domain of about residues 1 -124 fused to a human immunoglobulin region.
  • Exemplary B7 and CD28 inhibitors include, but are not limited to, CTLA4, CTLA4-Fc, soluble forms of B7.1 , soluble forms of B7.2, an antagonist CD28 antibody, and an antagonist B7 antibody.
  • therapies comprise at least one of ICOS and B7RP1.
  • Certain examples include, but are not limited to, soluble forms of ICOS.
  • B7RP1 and ICOS sequences are disclosed in PCT published Application No. WO 00/46240.
  • therapies comprise at least one of CD40 and CD40L.
  • Certain examples include, but are not limited to, soluble forms of CD40.
  • the CD40L sequence is disclosed in WO 93/08207 and U.S. Patent No. 5,981 ,724.
  • the CD40 sequence is disclosed in Stamenkovic et al. EMBO J. 8, 1403 (1989).
  • CD40 Abs including antagonist Abs
  • CD40 ligand Abs including antagonist Abs
  • therapies comprise at least one of CD30 and CD30L.
  • the sequence of human CD30 is disclosed in Durkop et al. Cell 68, 421 (1992).
  • the sequence of human CD30 ligand is disclosed in W093/24135.
  • therapies comprise at least one of CD27 and CD27L.
  • the sequence of human CD27 is disclosed in Camerini et al. J. Immunol. 147, 3165 (1991).
  • the sequence of human CD27 ligand is disclosed in WO94/05691.
  • therapies comprise at least one of OX40 and OX40L.
  • Certain examples include soluble forms of OX40.
  • the sequence of human OX40 ligand is disclosed in Miura et al. Mol. Cell BioL 11 , 1313 (1991).
  • therapies comprise at least one of 4-1 -BB and 4-1 -BB ligand.
  • Certain examples include soluble forms of 4-1- BB.
  • the structures of 4-1 -BB and 4-1 -BB ligand and certain inhibitors thereof, are disclosed in WO 94/26290 and U.S. Patent Nos. 5,674,704 and 6,355,779.
  • therapies comprise at least one of TACI, BAFFR and AGP3.
  • Certain examples include, but are not limited to, soluble forms of TACI, BAFFR, AGP3 peptibody (an AGP3 tandem dimer peptide-Fc fusion), and anti-BlyS antibody.
  • AGP3 peptibody is disclosed in WO 02/92620.
  • Information on AGP3, TACI and BAFFR is available in WO 01/87977.
  • the sequence of human TACI is disclosed in W098/39361.
  • the ligand for TACI and methods for screening for inhibitors of the interaction of TACI with its ligand are disclosed in WO 00/67034.
  • AGP-3, TACI, and BAFFR, and inhibitors thereof are described, e.g., in WO 00/47740, WO 01/85782, WO 02/15273, WO 98/39361 , and von Bul ⁇ w and Bram (1997) Science 278:138-140. ,'
  • Exemplary TACI, BAFFR, and AGP3 inhibitors include, but are not limited to, soluble forms of TACI, soluble forms of BAFFR, AGP3 peptibody, anti-BlyS antibody, TACI-Fc, anti-BLIS antibody, BCMA-Fc, and BAFFR-Fc.
  • therapies comprise at least one of PD-1 and PD-1 L PD-1 is described in WO 93/08207. PD-1 L is described in WO 01/39722. [0267] In certain embodiments, therapies comprise an anti-CD20 antibody.
  • therapies comprise any of one or more slow-acting antirheumatic drugs (SAARDs) or disease modifying antirheumatic drugs (DMARDS), prodrug esters or pharmaceutically acceptable salts thereof for the treatment of of an inflammatory or autoimmune condition, as defined above, including, but not limited to, acute and chronic inflammation such as rheumatic diseases (e.g., lyme disease, juvenile (rheumatoid) arthritis, osteoarthritis, psoriatic arthritis, rheumatoid arthritis and staphylococcal-induced (“septic”) arthritis); and multiple sclerosis.
  • SAARDs slow-acting antirheumatic drugs
  • DARDS disease modifying antirheumatic drugs
  • prodrug esters or pharmaceutically acceptable salts thereof for the treatment of of an inflammatory or autoimmune condition, as defined above, including, but not limited to, acute and chronic inflammation such as rheumatic diseases (e.g., lyme disease, juvenile (rheumatoid) arthritis, osteoarthriti
  • SAARDs or DMARDS, prodrug esters and pharmaceutically acceptable salts thereof include, but are not limited to, allocupreide sodium, auranofin, aurothioglucose, aurothioglycanide, azathioprine, brequinar sodium, bucillamine, calcium 3-aurothio-2-propanol-1-sulfonate, chlorambucil, chloroquine, clobuzarit, cuproxoline, cyclophosphamide, cyclosporin, dapsone, 15-deoxyspergualin, diacerein, glucosamine, gold salts (e.g., cycloquine gold salt, gold sodium thiomalate, gold sodium thiosulfate), hydroxychloroquine, hydroxyurea, kebuzone, levamisole, lobenzarit, melittin, 6-mercaptopurine, methotrexate, mizoribine, mycophenolate
  • Methotrexate is an anti-metabolite and immunosuppressive drug.
  • methotrexate is an effective anti-inflammatory agent with utility in the treatment of severe and disabling psoriasis and rheumatoid arthritis (Hoffmeister (1983), The American Journal of Medicine, 30:69-73 and Jaffe (1988), Arthritis and Rheumatism, 31:299).
  • Methotrexate is N-[4-[(2,4-diamino-6- pteridinyl)methylamino]benzoyl]-L-glutamic acid and has the structural formula:
  • methotrexate Various activities of methotrexate have been demonstrated which likely contribute to its efficacy (Segal et al. (1990), Seminars in Arthritis and Rheumatism, 20: 190-198). The following mechanisms of action for methotrexate have been postulated: inhibition of folate-dependent pathways and protein metabolism (Morgan et al. (1987), Arthritis and Rheumatism, 30:1348-1356); inhibition of neutrophil migration into arthritic joints (Van de Kerkhof et al. (1985), British Journal of Dermatology, 113:251-255; Temowitz et al.
  • methotrexate may inhibit the proliferative effects of IL-1 and decrease monocyte IL-1 production in the short term in certain patients, this effect is not sustained and is unlikely to explain the long-term efficacy of methotrexate (Barrera et al. (1996), Seminars in Arthritis and Rheumatism, 25(4 ⁇ :234-253).
  • methotrexate is administered in combination with one or more compounds, including, but not limited to, an IL- 1 inhibitor, a TNF inhibitor, an IFN- ⁇ inhibitor, an IL-18 inhibitor, an inhibitor of the B7/CD28 pathway, an inhibitor of the ICOS/B7RP1 pathway, an inhibitor of the CD40 pathway, an inhibitor of the CD30 pathway, an inhibitor of the CD27 pathway, an inhibitor of the OX40 pathway, an inhibitor of the 4-1 -BB pathway, an inhibitor of the TACI, BAFFR, AGP3 pathway, an inhibitor of the PD-1 pathway, an inhibitor of the CD20 pathway, and other compounds that may be used to treat inflammation or an autoimmune condition.
  • compounds including, but not limited to, an IL- 1 inhibitor, a TNF inhibitor, an IFN- ⁇ inhibitor, an IL-18 inhibitor, an inhibitor of the B7/CD28 pathway, an inhibitor of the ICOS/B7RP1 pathway, an inhibitor of the CD40 pathway, an inhibitor of the CD30 pathway, an inhibitor of the CD27 pathway, an inhibitor
  • methotrexate is administered orally, intraperitoneally, subcutaneously, or intravenously.
  • a human patient may be treated with a combination of methotrexate and CTLA4-Fc or other B7/CD28 pathway inhibitor.
  • the patient takes a tablet or capsule of methotrexate three times a week, at a total weekly dose of 5 to 50 mg/week.
  • the patient is injected intravenously with CTLA4-Fc at a daily dose between 0.1 mg/kg and 100 mg/kg.
  • the starting doses of the particular compounds used are reduced for a patient who exhibits an adverse reaction.
  • one or more of the drugs used in the combination can be changed or reduced, e.g., depending on the different formulations, routes, dose schedules, or other variables known to those skilled in the art, including, but not limited to, the individual patient's tolerance of the drug, its efficacy, and toxicity.
  • the patient is treated with a weekly starting dose of methotrexate at between 5 mg and 7.5 mg (orally or intramuscularly), and a daily dose of CTLA4-Fc at between 0.1 mg/kg and 100 mg/kg intravenously.
  • the dosage of methotrexate is increased by 5 mg every 2 to 3 weeks.
  • the maximum dosage level is determined at a point at which the patient shows improvements, which is generally less than about 25 mg of methotrexate per week, and, in certain embodiments, between 5 to 25 mg of methotrexate per week.
  • the evaluation includes physical examination and extensive laboratory testing.
  • the tests include evaluation for toxicity.
  • additional laboratory monitoring in the case of methotrexate includes a complete blood cell count every 2 weeks for the first 3 months and th ⁇ n monthly thereafter.
  • additional precautions include monthly assessments of levels of serum albumin, alanine amino transferase, bilirubin, creatinine, and blood urea nitrogen.
  • monthly urinalysis is performed. ,
  • methods of treating inflammatory and/or autoimmune diseases are provided.
  • pharmaceutical compositions are provided.
  • the co-stimulatory factors may be used in conjunction with cytokine inhibitors.
  • cytokine inhibitors of interest include, but are not limited to, IL-1 inhibitors and TNF- ⁇ inhibitors.
  • methods are provided for treating an IL-1 mediated disease, which comprises administering a therapeutically effective amount of an IL-1 inhibitor and an inhibitor of T cell or B cell activation.
  • methods are provided for treating a TNF- ⁇ mediated disease, which comprises administering a therapeutically effective amount of a TNF- ⁇ inhibitor and an inhibitor of T cell or B cell activation.
  • methods are provided for treating an inflammatory or an autoimmune condition, which comprises administering a therapeutically effective amount of an IL-1 inhibitor, a therapeutically effective amount of a TNF- ⁇ inhibitor, and an inhibitor of T cell or B cell activation.
  • Rheumatoid arthritis is a disease of unknown etiology that involves several components of the inflammatory process.
  • Clinical and experimental studies have shown that immune cells (T and B) along with proinflammatory cytokines create an imbalance in the biological system to cause local inflammation in the joints. Inhibition of IL-1 (by IL-1 Ra or KineretTM) or TNF- ⁇ (by soluble TNFR1 , TNFR2 or anti- TNF- ⁇ antibody) has been shown to be beneficial in experimental models of arthritis and in RA patients.
  • Clinical and experimental studies have also demonstrated the presence of T cells and B cells at the affected tissue.
  • Inhibitors of T cells and B cells have shown efficacy in experimental animal models when used from the induction of arthritis. Studies have shown that CTLA-4-lg is not efficacious in experimental animal models (Collagen-induced arthritis), when treatment starts from the clinical evidence of inflammatory arthritis.
  • a role for combination therapy that modulates T cell or B cell function with anti-proinflammatory cytokine medication in a model of experimental arthritis is discussed in Examples 1 to 3.
  • T cells, B cells and proinflammatory cytokines are involved in collagen-induced arthritis in mice.
  • Loeffler's syndrome chronic eosinophilic pneumonia, interstitial lung disease (ILD), such as idiopathic pulmonary fibrosis or ILD associated with rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, systemic sclerosis, Sjogren's syndrome, polymyositis or dermatomyositis); systemic anaphylaxis or hypersensitivity responses; drug allergy; insect sting allergy; inflammatory bowel disease, such as Crohn's disease and ulcerative colitis; spondyloarthropathy; scleroderma; psoriasis; inflammatory dermatosis such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria, vasculitis (e.g.
  • ILD interstitial lung disease
  • ILD interstitial lung disease
  • ILD interstitial lung disease
  • ILD inter
  • autoimmune diseases such as rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, juvenile onset diabetes, glomerulonephritis, autoimmune thyroiditis and Behcet's disease
  • graft rejection including allograft rejection or graft-versus-host disease
  • cancers with leukocyte infiltration of the skin or organs reperfusion injury; atherosclerosis; certain haematologic malignancies
  • shock including septic shock and endotoxic shock.
  • gene therapy may be employed.
  • a gene (either genomic DNA,' cDNA, and/or synthetic DNA) encoding a specific binding partner of a target molecule, or a fragment, variant, or derivative thereof, may be operably linked to a constitutive or inducible promoter to form a "gene therapy DNA construct".
  • the promoter may be homologous or heterologous to the gene encoding the specific binding partner, provided that it is active in the cell or tissue type into which the construct will be inserted.
  • Other components of a gene therapy DNA construct may include, but are not limited to, DNA molecules designed for site-specific integration (e.g., endogenous flanking sequences useful for homologous recombination), tissue-specific promoter, enhancer(s) or silencer(s), DNA molecules capable of providing a selective advantage over the parent cell, DNA molecules useful as labels to identify transformed cells, negative selection systems, cell specific binding agents (as, for example, for cell targeting) cell-specific internalization factors, and transcription factors to enhance expression by a vector as well as factors to enable vector manufacture.
  • DNA molecules designed for site-specific integration e.g., endogenous flanking sequences useful for homologous recombination
  • tissue-specific promoter e.g., enhancer(s) or silencer(s)
  • DNA molecules capable of providing a selective advantage over the parent cell DNA molecules useful as labels to identify transformed cells
  • negative selection systems e.g., cell specific binding agents (as, for example, for cell targeting) cell-specific internalization factors, and transcription factors to
  • a gene therapy DNA construct may then be introduced into a patient's cells (either ex vivo or in vivo).
  • the gene therapy DNA construct may be introduced via viral vectors.
  • Exemplary viral vectors used for delivery of gene therapy DNA constructs include, but are not limited to, adenovirus, adeno-associated virus, herpes simplex virus, lentivirus, papilloma virus, and retrovirus vectors.
  • some of these vectors, such as retroviral vectors will deliver the gene therapy DNA construct to the chromosomal DNA of the patient's cells, and the gene therapy DNA construct can integrate into the chromosomal DNA.
  • other vectors will function as episomes and the gene therapy DNA construct will remain in the cytoplasm.
  • the use of gene therapy vectors is described, for example, in U.S. Patent Nos. 5,672,344, 5,399,346.
  • Exemplary methods to deliver gene therapy DNA constructs to a patient's cells without the use of viral vectors include, but are not limited to, liposome-mediated transfer, direct injection of naked DNA, receptor-mediated transfer (ligand-DNA complex), electroporation, calcium phosphate precipitation, and microparticle bombardment (e.g., "gene gun”). See U.S. Patent Nos. 4,970,154, WO 96/40958, 5,679,559, 5,676,954, and 5,593,875.
  • the levels of expression of a specific binding partner in a cell may be increased via gene therapy by insertion of one or more enhancer elements into the promoter.
  • the enhancer elements used may be selected based on the tissue in which one desires to activate the genes, and enhancer elements known to confer promoter activation in that tissue will be selected.
  • the lck promoter enhancer element may be used to increase expression of a specific binding partner to be expressed in T-cells.
  • the functional portion of the transcriptional element to be added may be inserted into a fragment of DNA containing a promoter for a specific binding partner using standard cloning techniques. This construct, known as a "homologous recombination construct" can then be introduced into the desired cells either ex vivo or in vivo.
  • gene therapy may be used to decrease expression of target molecules by modifying the nucleotide sequence of the endogenous promoter(s). In certain embodiments, the modification may be accomplished via homologous recombination methods.
  • a DNA molecule containing all or a portion of the promoter of a gene encoding a target molecule may be engineered to remove and/or replace pieces of the promoter that regulate transcription.
  • the TATA box and/or the binding site of a transcriptional activator of the promoter may be deleted using standard molecular biology techniques; such deletion can inhibit promoter activity thereby repressing transcription of the corresponding target molecule gene.
  • deletion of the TATA box or transcription activator binding site in a promoter may be accomplished by generating a DNA construct comprising all or the relevant portion of a promoter in Which one or more of the TATA box or transcriptional activator binding site hucleotides are mutated via substitution, deletion and/or insertion of one or more nucleotides such that the TATA box or activator binding site has decreased activity or is rendered completely inactive.
  • this construct may contain at least about 500 bases of DNA that correspond to the native (endogenous) 5' and 3' flanking regions of the promoter segment that has been modified, and may be introduced into the appropriate cells (either ex vivo or in vivo) either directly or via a viral vector as described above.
  • integration of the construct into the genomic DNA of the cells may be via homologous recombination, where the 5' and 3' flanking DNA sequences in the promoter construct can serve to help integrate the modified promoter region via hybridization to the endogenous chromosomal DNA.
  • antisense DNA or RNA molecules which have a sequence that is complementary to at least a portion of a selected target molecule gene may be introduced into the cell.
  • each such antisense molecule will be complementary to the start site (5' end) of each selected target molecule gene.
  • gene therapy may be employed to create a dominant-negative inhibitor of one or more target molecules.
  • the DNA encoding a mutant full length or truncated polypeptide of each selected target molecule can be prepared and introduced into the cells of a patient using either viral or non-viral methods as described above.
  • each mutant may be designed to compete with an endogenous target molecule in its biological role.
  • the invention provides for pharmaceutical compositions comprising a therapeutically effective amount of certain particular molecules together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.
  • the invention provides for pharmaceutical compositions comprising certain particular molecules, together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.
  • acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.
  • the pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition.
  • suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris- HCI, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents;
  • amino acids
  • one or more of the molecules are linked to a half-life extending vehicle known in the art.
  • a half-life extending vehicle known in the art.
  • Such vehicles include, but are not limited to, the Fc domain, polyethylene glycol, and dextran.
  • Such vehicles are described, e.g., in published PCT Application No. WO 00/24782, which is hereby incorporated by reference for any purpose.
  • the optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of the antibodies of the invention.
  • the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
  • neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor.
  • a composition comprising certain particular molecules may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, in certain embodiments, a composition comprising certain particular molecules may be formulated as a lyophilizate using appropriate excipients such as sucrose.
  • the pharmaceutical compositions of the invention can be selected for parenteral delivery.
  • the compositions may be selected for inhalation or for delivery through the digestive tract, such as orally.
  • the preparation of such pharmaceutically acceptable compositions is within the skill of the art.
  • the formulation components are present in concentrations that are acceptable to the site of administration.
  • buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
  • a therapeutic composition when parenteral administration is contemplated, may be in the form of a pyrogen- free, parenterally acceptable aqueous solution comprising the desired certain particular molecules in a pharmaceutically acceptable vehicle.
  • a vehicle for parenteral injection is sterile distilled water in which certain particular molecules are formulated as a sterile, isotonic solution, properly preserved.
  • the preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide for the controlled or sustained release of the product which may then be delivered via a depot injection.
  • hyaluronic acid may also be used, and may have the effect of promoting sustained duration in the circulation.
  • implantable drug delivery devices may be used to introduce the desired molecule.
  • a pharmaceutical composition may be formulated for inhalation.
  • certain particular molecules may be formulated as a dry powder for inhalation.
  • an inhalation solution comprising certain particular molecules may be formulated with a propellant for aerosol delivery.
  • solutions may be nebulized.
  • Pulmonary administration is further described in PCT application no. PCT/US94/001875, which describes pulmonary delivery of chemically modified proteins.
  • formulations may be administered orally.
  • certain particular molecules that are administered in this fashion may be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
  • a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized.
  • at least one additional agent can be included to facilitate absorption of certain particular molecules.
  • diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.
  • a pharmaceutical composition may involve an effective quantity of certain particular molecules in a mixture with non-toxic excipients which are suitable for the manufacture of tablets.
  • suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
  • sustained-release preparations may include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules.
  • Sustained release matrices may include polyesters, hydrogels, polylactides (U.S. 3,773,919 and EP 058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman etal., Biopolymers, 22:547-556 (1983)), poly (2-hydroxyethyl- methacrylate) (Langer et al., J. Biomed. Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., supra) or poly-D(-)-3-hydroxybutyric acid (EP 133,988).
  • sustained release compositions may also include liposomes, which can be prepared by any of several methods known in the art. See e.g., Eppstein et al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP 088,046 and EP 143,949.
  • the pharmaceutical composition to be used for in vivo administration typically is sterile. In certain embodiments, this may be accomplished by filtration through sterile filtration membranes. In certain embodiments, where the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution. In certain embodiments, the composition for parenteral administration may be stored in lyophilized form or in a solution. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
  • the pharmaceutical composition may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
  • such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration.
  • kits for producing a single-dose administration unit may each contain both a first container having a dried protein and a second container having an aqueous formulation.
  • kits containing single and multi-chambered pre-filled syringes e.g., liquid syringes and lyosyringes are included.
  • the effective amount of a pharmaceutical composition comprising certain particular molecules to be employed therapeutically will depend, for example, upon the therapeutic context and objectives.
  • the appropriate dosage levels for treatment will thus vary depending, in part, upon the molecule delivered, the indication for which certain particular molecules are being used, the route of administration, and the size (body weight, body surface or organ size) and/or condition (the age and general health) of the patient.
  • the clinician may titer the dosage and modify the route of administration to obtain the optimal therapeutic effect.
  • a typical dosage may range from about 0.1 ⁇ g/kg to up to about 100 mg/kg or more, depending on the factors mentioned above.
  • the dosage may range from 0.1 ⁇ g/kg up to about 100 mg/kg; or 1 ⁇ g/kg up to about 100 mg/kg; or 5 ⁇ g/kg up to about 100 mg/kg.
  • the frequency of dosing will take into account the pharmacokinetic parameters of certain particular molecules in the formulation used.
  • a clinician will administer the composition until a dosage is reached that achieves the desired effect.
  • the composition may therefore be administered as a single dose, or as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them.
  • appropriate dosages may be ascertained through use of appropriate dose-response data.
  • the route of administration of the pharmaceutical composition is in accord with known methods, e.g. orally, through injection by intravenous, intraperitoneal, intracerebral (intra- parenchymal), intracerebroventricular, intramuscular, intra-ocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices.
  • the compositions may be administered by bolus injection or continuously by infusion, or by implantation device.
  • the composition may be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.
  • a pharmaceutical composition comprising certain particular molecules in an ex vivo manner.
  • cells, tissues and/or organs that have been removed from the patient are exposed to a pharmaceutical composition comprising certain particular molecules after which the cells, tissues and/or organs are subsequently implanted back into the patient.
  • certain particular molecules can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the polypeptides.
  • such cells may be animal or human cells, and may be autologous, heterologous 1 , or xenogeneic.
  • the cells may be immortalized.
  • the cells in order to decrease the chance of an immunological response, the cells may be encapsulated to avoid infiltration of surrounding tissues.
  • the encapsulation materials are typically biocompatible, semipermeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues. Examples
  • murine CTLA4-Fc was derived from murine CTLA4 (SEQ ID NO. 19).
  • Freund's Complete Adjuvant (CFA) was obtained from Difco (Detroit, Ml).
  • immunization grade porcine type II collagen was purchased from Griffith's lab (University of Utah, Salt Lake City).
  • Murine CTLA4-Fc and KIN2 (SEQ ID NO 2) used in the study were produced at Amgen. A. Exemplary preparation of KIN2
  • KIN2 may be produced as generally described in U.S.
  • Patent No. 6,294,170 B1 (the '170 patent).
  • the Fc-IL-1 ra fusion protein discussed in the '170 patent is the same as KIN2 discussed herein.
  • the '170 patent discusses an IL-1 ra gene fragment being enzymatically cleaved from another expression vector and being ligated to the expression vector pAMG21 (European Patent Application No. 96309363.8).
  • amino acid sequence of IL-1 ra is:
  • the '170 patent discusses that the resulting plasmid pAMG21-IL-1 ra was purified and the sequence of the IL-1 ra gene was confirmed by sequencing.
  • the '170 patent discusses that that plasmid (pAMG21-IL-1 ra)(European Patent Application No. 96309363.8) was used later for cloning of rhulL-1 ra-Fc protein.
  • the '170 patent states that an rhulL- 1 ra fusion protein was constructed where the Fc region of human lgG1 was fused at the N-terminus of human IL-1 ra.
  • the Fc sequence that was chosen for fusions in the '170 patent is shown below. Eight extra amino acid residues AAAEPKSS are present in the N-terminus of the functional Fc region.
  • W1485 (a K12 strain) was obtained from the E. coli Genetic Stock Center, Yale University, New Haven, CT (CGSC strain #6159). The strain is prototrophic, contains no lambda prophage, and has been cured of the sex factor, F.
  • the '170 patent states that the CGSC strain #6159 has been altered by selecting for spontaneous resistance to four different phages isolated from phage outbreaks that occurred while conducting fermentation research.
  • the '170 patent states that a first round of phage-resistant mutant isolation, conferring resistance simultaneously to two of the four phages, was performed.
  • the '170 patent states that a sample of one of the phages was diluted and mixed with a culture of the sensitive strain and incubated as a liquid culture at 37°C for 16-24 hours to select for phage-resistant survivors.
  • the '170 patent states that candidates were isolated from single colonies and tested to confirm phage resistance and ability to grow in minimal medium.
  • the '170 patent states that the mutation obtained in the first round of selection exhibits certain characteristics of a tonA mutation in that the strain simultaneously acquired resistance to phages T5 and ⁇ 80.
  • the '170 patent states that a second phage resistance selection, conferring resistance to the third phage, was performed on May 15, 1984.
  • the '170 patent states that spontaneous phage-resistant mutants were obtained using a plate method. Lawns of sensitive bacteria were spotted with a phage suspension and incubated at 37°C for two days. Survivors were isolated from colonies in the zone of lysis. They were tested for growth in minimal medium, normal efficiency of plasmid transformation, normal growth rate in complex medium, and normal level of product synthesis.
  • the '170 patent states that the mutation conferring resistance to this phage has been mapped at the btu locus of E. coli.
  • the '170 patent also discusses a third round of phage resistance selection performed using the plate method described above.
  • the '170 patent states that the purified mutant appeared normal by those criteria outlined above (growth in minimal and complex media, efficiency of transformation and level of product synthesis).
  • the '170 patent states that the unique Sad I site in the Fc region and the unique Sacl site in the IL-1 ra gene were used for cloning.
  • the Sacll-Sacl fragment was synthesized using standard PCR technology. Templates for PCR reactions were plasmid preparations (pAMG21-OPG-Fc and pAMG21-IL-1 ra) containing the target genes.
  • the '170 patent states that the overlapping oligos were designed to combine the C-terminal portion of the Fc gene with the N terminal portion of the IL-1 ra gene.
  • the '170 patent states that this process allows fusing the two genes together in the correct reading frame after the appropriate PCR reactions have been performed.
  • the '170 patent states that the final PCR gene products were digested with restriction endonucleases Sacll and Sacl, and a three-way ligation was conducted with the Clal-Sacll Fc fragment with partial pAMG21 sequence isolated from pAMG21-Fc-OPG and the vector Clal-Sacl fragment with partial IL-1 ra sequence isolated from pAMG21-IL-1 ra.
  • the ligation mixture was transformed into E. coli host by electroporation utilizing the manufacturer's protocol.
  • the '170 patent states that clones were screened for the ability to produce the recombinant Fc-rhulL-1 ra and to possess the gene fusion having the correct nucleotide sequence. A methionine residue was added to the junction of the Fc region and the rhulL-1 ra, but it did not interfere with the activity of the fusion protein.
  • Fc-IL-1 ra fusion protein was placed under control of the luxPR promoter in pAMG21 (U.S. Patent No. 5,169,318 for description of the lux expression system).
  • the '170 patent states that cultures of pAMG21 -Fc-IL-1 ra and E. coli host were placed in Terrific broth media (Tartof. and Hobbs (1987), Bethesda Res. Lab. Focus, 9:12) containing 50 ⁇ g/ml kanamycin and were incubated at 30°C to an OD600 of about 0.8 prior to induction.
  • Inclusion bodies (IB) may be mixed at a concentration of
  • the solubilized inclusion bodies may be slowly, drop by drop, diluted to a concentration of 1 :20 (vol./vol.) into the refolding buffer of 2.5 M 1 ,3-Dimethyl-Urea, 0.2 M Arg, 4.5 mM cysteine and 1 mM cystamine dihydrochloride pH8.8.
  • the mixture may then set at 4°C overnight, without stirring.
  • the refolded mixture may be centrifuged for 1 hour in a
  • the resultant supernatant may be concentrated 3 times and buffer changed into 25mM tris pH 8.8 with an ultrafiltration system.
  • the retentate may be titrated by 6M HCI to pH 6.5. Some precipitate may be . formed at pH 6.5 and may be discarded after being centrifuged for 1 hour in the J6-B centrifuge.
  • the supernatant from ultracentrifugation may be passed through a column of Q High Performance (QHP) (15 g IB's/50ml) resin (Pharmacia Biotech, Inc., Piscataway, NJ) pre-equilibrated with 20mM 2-n- morpholinoephane-sulfonic acid pH 6.5 buffer.
  • QHP Q High Performance
  • the flow-through from the QHP (QFT) and the early eluted fractions from the QHP column which contains the product may be collected.
  • Solid ammonium sulfate may be added into the Q-FT pool to a concentration of 0.7 M.
  • the sample may then be loaded onto a Phenyl-toyo column (Toso Haas, Philadelphia, PA) (15g IB's/15 ml resin) pre- equilibrated with 0.7 M ammonium sulfate in 20 mM sodium phosphate pH 7.0. After washing with 0.7 M ammonium sulfate, the column may be eluted with a linear gradient from 0.7 M ammonium sulfate to 0.2 M ammonium sulfate in 20 mM sodium phosphate pH 7.0 buffer. Fractions containing the product may be pooled based on SDS-PAGE analysis.
  • the HIC pool may be dialyzed info 25 mM sodium phosphate pH 7.0.
  • the dialyzed sample may be titrated by 2 M citric acid to pH 5.0, and then loaded onto a SOHO column (15 g IB's/7 ml resin) pre- equilibrated with 20 mM citric acid pH 5.0. After washing with 25 mM citric acid pH 5.0 and pH 6.0 buffer the column may be eluted with a linear gradient from 0 M to 0.7 M NaCl in 25 mM citric acid pH 6.0 buffer.
  • the fractions containing the product may be pooled.
  • Porcine type II collagen was dissolved in 0.01 N acetic acid at a concentration of 2 mg/ml and then emulsified at a 1 :1 ratio with CFA (Difco).
  • Arthritis susceptible B10.RIII (H-2 r ) mice from Jackson Lab, Bar harbor, Maine, USA) were immunized with 100 ⁇ l of emulsion intradermally at the base of the tail. (Nabozny et al. Autoimmunity 20, 39-49 (1995)).
  • mice were monitored once every day for the development of arthritis. Arthritis severity was determined using a grading system (Khare et al. J. Immunol., 155: 3653-3659, 1995) for each paw as follows: 0: no arthritis; 1 : redness or swelling 1-3 toes; 2: severe swelling of paw; 3: joint ankylosis. The score of each limb was summed, thus giving a severity range from 0 to 12 for each animal. The scores for the mice were summed and divided by the total number of mice to generate a mean arthritis score. As mice developed disease, they were randomized to study groups (9-10 mice/group) and treatment was initiated.
  • KIN2 was suspended in A5S (A5S buffer includes 10mM Acetic Acid and 5% Sorbitol pH 5.0) and murine CTLA4-Fc was suspended in phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Mice were treated by injection of 100 ⁇ g of KIN2, 100 ⁇ g murine CTLA4-Fc, or 100 ⁇ g each of KIN2 and murine CTLA4-Fc at day 0, +1 , +2, +4, +6, +8 and +10 post clinical onset of disease. 100 ⁇ g is equivalent to 5 mg of therapeutic per Kg weight of animal (5mg/Kg).
  • Mice were injected in 100 ⁇ l volumes, except in the combination experiment, wherein the mice received two separate injections of 100 ⁇ l each. Injections of PBS were used as a control. Mice were monitored once every day and a mean arthritis score was calculated.
  • KIN2 reduced the severity of collagen-induced arthritis in mice when given alone whereas murine CTLA4-Fc given alone had no effect when compared to PBS control.
  • the combination of KIN2 and murine CTLA4-Fc reduced collagen-induced arthritis for an extended period of time compared to KIN2 alone, as shown in Figure 1.
  • PEG sTNFR-1 2.6D, murine CTLA4-Fc, and Fc used in the study were produced at Amgen.
  • sTNFR-1 2.6D molecules include, but are not limited to, sTNFR-1 2.6D/C105, sTNFR-1 2.6D/C106, and sTNFR-1 2.6D/N105 (SEQ ID NO. 4).
  • PEG may be added to a sTNFR-1 2.6D molecule in a manner similar to how PEG may be added to sTNFR-1 2.6D/N105, as generally described in published PCT Application No. WO 98/01555 (PCT '555).
  • PCT '555 Exemplary Preparation of sTNFR-l 2.6D/N105-t-BuPEG(33kDa, in PCT '555
  • NaCNBH3 is added to a final
  • PCT '555 states that the extent of the protein modification during the course of the reaction is monitored by SEC HPLC using a TSKG3000SWXL column (Toso Haas, Montgomeryville, PA) eluting with 0.1 M sodium phosphate buffer pH 6.9, 0.5M NaCl, and 10% ethanol at 0.7 ml/min (Toso Haas, Montgomeryville, PA).
  • PCT '555 states that the pH of the reaction mixture is adjusted to ca. 3.5 with 1 M HCI, and the reaction mixture is diluted with water to a final protein concentration of 1.5 mg/ml.
  • PCT '555 states that sTNFR-l 2.6D/N105-t-
  • BuPEG(33kDa) is separated from the excess of t-BuPEG and other reaction by-products by using a SP Sepharose HP 16/10TM ion-exchange chromatography (Pharmacia Biotech, Inc., Piscataway, NJ).
  • PCT '555 states that the reaction mixture is loaded onto the column and the unreacted t-BuPEG is eluted with 3 column volumes of the starting Buffer A (20mM sodium acetate, pH 4.0).
  • the sTNFR-l 2.6D/N105-t-BuPEG(33kDa) is eluted using a linear 20 column volume gradient from 0-30% Buffer B (1 M NaCl in 20mM acetate, pH 4.0.
  • the eluent is monitored at 280 nm.
  • Each fraction containing sTNFR-l 2.6D/N105-t- BuPEG(33kDa) is analyzed by SDS-PAGE using 4-20% precast gradient gels (Novex, San Diego, CA).
  • PCT '555 states that to a cooled (7 °C), stirred solution of
  • STNFR-2.6D/N105 (4 mg/ml) is added 10% acetic acid until the pH is 5.0.
  • PCT '555 states that after 18 hours, the protein concentration in the reaction mixture is adjusted to pH 3.0 with citric acid.
  • PCT '555 states that sTNFR-l 2.6D/N105-MePEG(33kDa) is separated from the excess of MePEG and other reaction by-products by ion exchange chromatography using an SP Sepharose HPTM column (Pharmacia Biotech, Inc., Piscataway, NJ).
  • PCT '555 states that the reaction mixture is loaded (no more than 8mg/ml of resin) onto the column and the unreacted MePEG is eluted with 3 column volumes of the starting buffer A (20mM sodium citrate, pH 3.0).
  • the sTNFR-l 2.6D/N105-MePEG(33kDa) is eluted using a linear 16 column volume gradient from 0.1 - 0.5. M NaCl in 20mM citrate, pH 3.0.
  • the eluent is monitored at 280 nm.
  • Each fraction containing sTNFR-l 2.6D/N105- MePEG(33kDa) is analyzed by SDS-PAGE using 4-20% precast gradient gels (Novex, San Diego, CA). Based on SDS-PAGE analysis results, fractions are pooled, concentrated, and sterile filtered. Each final pool of purified sTNFR-l 2.6D/N105-MePEG(33kDa) is again analyzed by SDS-PAGE.
  • the purified sTNFR-l 2.6D/N105-MePEG(33kDa) is concentrated to 5 - 20 mg/mL and formulated in either PBS, pH 6.5 (10 mM sodium phosphate, 35-100 mM NaCl) or 20mM acetate, 100 mM NaCl, pH 5.0.
  • PBS pH 6.5
  • 20mM acetate 100 mM NaCl, pH 5.0.
  • PEG sTNFR-1 2.6D reduced the severity of collagen- induced arthritis in mice when given alone whereas m ⁇ rine CTLA4-Fc given alone had no effect when compared to PBS control.
  • the combination of PEG sTNFR-1 2.6D and murine CTLA4-Fc reduced collagen-induced arthritis for an extended period of time compared to PEG sTNFR-1 2.6D alone, as shown in Figure 2.
  • Serum samples were taken ten days after the conclusion of the experiment shown in Figure 2 and assayed for type II collagen specific antibodies. Briefly, porcine type II collagen at 2 ⁇ g/ml was coated onto high- binding plates (Nalge Nunc Intl., Denmark) overnight. The plates were washed with PBS-Tween 20 and blocked with 1% bovine serum albumin (BSA) in PBS. Serum samples were added to the plates at dilutions ranging from 1 :100 to 1 :6400, and incubated for three hours at room temperature. After incubation, excess sera was washed from the plates with PBS-Tween 20.
  • BSA bovine serum albumin
  • Anti-lgG1 and anti-lgG2b antibodies (Southern Biotech, Birmingham, AL) labeled with Horse Radish Peroxidase (HRP) were added to the plates and incubated for 1 to 2 hours. Excess reagents were washed from the plates with PBS-Tween 20. The reaction was developed using a 3,3',5,5'- Tetramethyl-Benzidine (TMB) solution (Sigma, St. Louis, MO) and the optical density at 450 nm was measured. The optical density values obtained were compared with standard serum from type II collagen immunized mice.
  • TMB 3,3',5,5'- Tetramethyl-Benzidine
  • the level of type II collagen specific antibodies was reduced in animals injected with PEG sTNFR-1 2.6D and murine CTLA4-Fc compared to animals injected with murine CTLA4-Fc alone as measured by anti-lgG1 antibodies, and shown in Figure 3A.
  • the level of type II collagen specific antibodies was reduced in animals injected with PEG sTNFR-1 2.6D and murine CTLA4-Fc compared to both animals injected with PEG sTNFR-1 2.6D alone and animals injected with murine CTLA4-Fc alone, as measured by anti-lgG2b antibodies, and shown in Figure 3B.
  • EXAMPLE 3 Combination therapy using viral vectors expressing IL-1 a, sTNFR-l and/or CTLA4
  • Adeno-associated virus (AAV) cloning vectors were based on the pBluescript SK II vector (Stratagene, La Jolla, CA). An oligonucleotide linker containing a Bgl II site
  • CGCGAGATCTTGCGCAAGATCT (SEQ ID NO. 28) was inserted between the two BssH II sites of pBSSK II.
  • AAV2 genomic DNA (available from the American Type Culture Collection under accession number 37215; see also Laughlin et al. Gene 23, 65-73 (1983)) having flanking Bgl II sites was inserted into the linker.
  • a Nrul site and a Xhol site were introduced by site directed mutagenesis at positions 145 and 4530 respectively of the AAV2 nucleotide sequence (Srivastava et. al. J. Virol. 45, 555-564 (1983) and NCBI Accession No. NC-001401).
  • the resulting construct was digested with Nrul and Xhol and the AAV2 sequence from nucleotide 146 to nucleotide 4534 was removed.
  • a DNA fragment comprising either a cytomegalovirus (CMV) early gene promoter/enhancer (Invitrogen, Carlsbad, CA) or an EF1a. promoter (PEFI HisA, Invitrogen) followed by a multiple cloning site derived from pcDNA3.1 (Invitrogen) was inserted between remaining nucleotides 145 and 4535 of the AAV2 sequence.
  • CMV cytomegalovirus
  • PFI HisA early gene promoter/enhancer
  • the bovine growth hormone poly adenylation site from pcDNA3.1 was inserted downstream of the multiple cloning site.
  • cDNA encoding either human IL-1 ra (SEQ ID NO. 20), rat sTNFR-1 , human CTLA4 (SEQ ID NO. 2), or beta-galactosidase was cloned into Nhe I and Not I sites respectively using standard cloning procedures.
  • Rat sTNFR-1 was derived from rat TNFR-I (SEQ ID NO. 21).
  • the packaging plasmid pTrans was constructed using the pBluescript SK+ vector (Stratagene, La Jolla, CA).
  • An AAV2 genomic fragment spanning nucleotides 191-4497 in the AAV2 genome was obtained by digestion with Clal and SnaBI and the fragment was inserted into pBluescript SK+ digested with Clal and EcoRV.
  • AAV2 nucleotide sequence 1849-2202 was inserted into AAV2 nucleotide sequence at nucleotide 320 using standard cloning methods.
  • An adenovirus helper plasmid containing the adenovirus E2a, E4orf 6 and VA sequence of adenovirus 5 was cloned into pBluescript SK+ using standard cloning procedures. /
  • Monolayers of human embryonic kidney 293T cells were cultured in 5% C0 2 at 37 °C in medium A (Pulbecco's modified Eagle's medium containing 100 units/ml penicillin and 100 ⁇ g/ml streptomycin sulfate) supplemented with 10% (v/v) fetal calf serum.
  • Cotransfection of AAV cloning vector, pTrans and pHelper plasmid in ratio of 1 :1 :3 were performed using a calcium phosphate precipitation method (Sambrook et al. Molecular Cloning, supra). Cells were harvested and viruses were released by freezing and thawing. Viruses were purified using heparin sulfate chromatography and titered using a dot blot method described in Auricchiv et. al. Human Gene Therapy 12,71 -76).
  • mice were injected intravenously with 100 ⁇ l of PBS containing 7 x 10 12 particles of recombinant AAV carrying ⁇ -gal, IL-1 ra, sTNFR-1 , CTLA-4 or a combination as indicated.
  • Blood was collected from tail veins for ELISA to quantify the protein expression level.
  • mice were boosted with 100 ⁇ g porcine type II collagen in 1x CFA intradermally.
  • CTLA4 reduced the incidence and severity of collagen-induced arthritis in mice when vectors were administered individually compared to a ⁇ - galactosidase control.
  • the combination of sTNFR-l and CTLA4 expression by coadministration of vectors reduced the severity and incidence of collagen- induced arthritis to a greater extent than expression of those proteins individually, as shown in Figures 4 and 5.
  • Serum samples from the treatment groups of mice shown in Figure 4 were taken several weeks after booster of porcine type II collagen and levels of type II collagen specific antibodies were assayed as described above in Example 2, except that anti-lgG3 antibodies were used.
  • the level of type II collagen specific antibodies was reduced in animals injected with sTNFR-1 and CTLA-4 vectors compared to animals injected with a sTNFR-1 vector alone as measured by anti-lgG3 antibodies, and shown in Figure 6.
  • the level of type II collagen specific antibodies was reduced in animals injected with IL1-ra and CTLA-4 vectors compared to both animals injected with an IL1-ra vector alone and animals injected with a CTLA-4 vector alone, as measured by anti-lgG3 antibodies, and shown in Figure 6.
  • CTLA4-Fc protein and the AGP3 peptibody an
  • AGP3 tandem dimer peptide-Fc fusion (“AGP3 Pb”)(SEQ IP NO. 1) were produced at Amgen.
  • AGP3 Pb may be prepared as generally described in published PCT Application No. WO 02/092620 (PCT '620).
  • PCT '620 discusses that an inhibitory AGP-3 Pb was constructed in which a tandem dimer of a peptide was fused in-frame to the Fc region of human lgG1.
  • PCT '620 states that an inhibitory peptibody may be constructed by annealing pairs of oligonucleotides to generate a duplex encoding the peptide and a linker comprised of 5 glycine residues and one valine residue as an Ndel to Sail fragment.
  • PCT '620 states that these duplex molecules were ligated into a vector (pAMG21-RANK-Fc, described in PCT '620) containing the human Fc gene, also digested with Ndel and Sail.
  • PCT '620 states that the resulting ligation mixtures were transformed by electroporation into E. coli strain 2596 cells (GM221 , described in PCT '620).
  • PCT '620 states that clones were screened for the ability to produce the recombinant protein product and to possess the gene fusion having the correct nucleotide sequence.
  • PCT '620 states that a single such clone was selected for each of the peptibodies.
  • pAMG21 -RANK-Fc fusion constructs may be expressed in E. coli as described in WO 02/092620.
  • mice spontaneously develop lupus-like symptoms at 6 to 9 months of age and usually die at 10 to 12 months of age (Vyse et al. Ann Rev Immunol 16, 261-292 (1998)).
  • mice Prior to treatment, mice were pre-screened for protein in the urine with Albustix® reagent strips (Bayer Corp., Elkhart, IN) and those having greater than 100 mg/dl of protein in the urine were not included in the experiment.
  • murine CTLA4-Fc and AGP3 Pb were suspended in PBS.
  • Serum samples taken from the various treatment groups of mice at day 0, 30, 60, and 90 were assayed for the presence of antibodies to double stranded PNA.
  • Adsorbent plates were coated overnight at 4 °C. with 50 ⁇ l of a 1 :1 solution of 1 ⁇ g/ml plasmid PNA (Immunovision, Springdale, AZ) diluted in PBS with 1 mg/ml methylated bovine serum albumin (BSA; Sigma, St. Louis, MO) dissolved in water. Plates were gently washed three times with PBS/Tween 20 and blocked with 75 ⁇ l of 2% BSA/PBS for up to 2 hours.
  • BSA methylated bovine serum albumin
  • PEG sTNFR-1 2.6D, AGP3 Pb (an AGP3 tandem dimer peptide-Fc fusion), and Fc used in the study were produced at Amgen.
  • Exemplary PEG sTNFR-1 2.6D production is described in Example 2.
  • Exemplary AGP3 Pb production is described in Example 4.
  • AGP3 Pb was suspended in PBS and sTNFR-1 2.6D was suspended in PBS.
  • Mice were administered by injection of 100 ⁇ g of PEG sTNFR-l 2.6D, 100 ⁇ g AGP3 Pb, or 100 ⁇ g each of PEG sTNFR-1 2.6Dand AGP3 Pb at day 0, +1 , +2, +4, +6, +8 and +10 post clinical onset of disease. 100 ⁇ g is equivalent to 5 mg of therapeutic per Kg weight of animal (5mg/Kg).
  • Mice were injected in 100 ⁇ l volumes.
  • PBS and human IgGI Fc Protein Science, Amgen, Thousand Oaks, CA
  • Mice were evaluated once a day for a mean arthritic score as described in Example 1.
  • AGP3 Pb an AGP3 tandem dimer peptide-Fc fusion
  • KIN2 used in the study were produced at Amgen. Exemplary KIN2 production is described in Example 1. Exemplary AGP3 Pb production is described in Example 4. Therapy in Mice
  • AGP3 Pb was suspended in PBS. Mice were treated by injection of 100 ⁇ g of KIN2, 100 ⁇ g AGP3 Pb, or 100 ⁇ g each of KIN2 and AGP3 Pb at day 0, +1 , +2, +4, +6, +8 and +10 post clinical onset of disease. 100 ⁇ g is equivalent to 5 mg of therapeutic per Kg weight of animal (5mg/Kg). Mice were injected in 100 ⁇ l volumes, except in the combination experiment, wherein the mice received two separate injections of 100 ⁇ l each. Injections of PBS and Fc were used as controls. Mice were monitored once every day and a mean arthritis score was calculated as described in Example 1.
  • KIN2 reduced the severity of collagen-induced arthritis in mice when given alone, whereas AGP3 Pb given alone had a minimal effect when compared to PBS control.
  • the combination of KIN2 and AGP3 Pb reduced collagen-induced arthritis for an extended period of time compared to KIN2 alone, as shown in Figure 13.
  • KIN2 used in the study was produced at Amgen.
  • Example 1 p Exemplary KIN2 production is described in Example 1 p .
  • Anti-OX40L antibody (RM134L) used in the study was purchased from Pharmingen (San Diego, CA). Exemplary generation of anti-OX40L antibody is summarized by Akiba et al. (J Immunol, 1999, 162: 7058-7066). Rat IgG was purchased from Sigma (St. Louis, MO).
  • DBA/1 mice were immunized with bovine type II collagen emulsified with 1X complete Freunds adjuvant. Three weeks later, non- arthritic mice were boosted with bovine type II collagen emulsified with incomplete Freunds adjuvant. Mice were monitored for the development of arthritis as described in Example 1.
  • OX40L antibody was suspended in PBS. Mice were treated by injection of 100 ⁇ g of KIN2, 100 ⁇ g anti-OX40L antibody, or 100 ⁇ g each of KIN2 and anti-OX40L antibody, or 100 ⁇ g each of KIN2 and anti-OX40L antibody at day 0, +1 , +2, +4, +6, +8 and +10 post clinical onset of disease. 100 ⁇ g is equivalent to 5 mg of therapeutic per Kg weight of animal (5mg/Kg). Mice were injected in 100 ⁇ l volumes, except in the combination experiments, wherein the mice received two separate injections of 100 ⁇ l each. Injections of PBS and Rat IgG were used as controls. Mice were monitored once every day and a mean arthritis score was calculated as described in Example 1.
  • KIN2 reduced the severity of collagen-induced arthritis in mice when given alone, whereas anti-OX40L antibody given alone had a minimal effect when compared to PBS control.
  • the combination of KIN2 and anti-OX40L antibody reduced collagen-induced arthritis for an extended period of time compared to KIN2 alone, as shown in Figure 14.
  • PEG sTNFR-1 2.6D and Fc used in the study were produced at Amgen. Exemplary PEG sTNFR-1 2.6D production is described in Example 2.
  • Anti-OX40L antibody was purchased from Pharmingen. Rat IgG was purchased from Sigma.
  • anti-OX40L antibody was suspended in PBS and PEG sTNFR-1 2.6D was suspended in PBS. Mice were administered by injection of 100 ⁇ g PEG sTNFR-1 2.6D, 100 ⁇ g anti-OX40L antibody, or 100 ⁇ g each of PEG sTNFR-1 2.6D and anti-OX40L antibody at day 0, +1 , +2, +4, +6, +8 and +10 post clinical onset of disease. 100 ⁇ g is equivalent to 5 mg of therapeutic per Kg weight of animal (5mg/Kg). Mice were injected in 100 ⁇ l volumes. Injections of PBS and Rat IgG were used as controls. Mice were evaluated once a day for a mean arthritic score as described in Example 1.
  • PEG sTNFR-1 2.6D reduced the severity of collagen- induced arthritis in mice when given alone, whereas anti-OX40L antibody given alone had a minimal effect when compared to PBS control.
  • the combination of PEG sTNFR-1 2.6D and anti-OX40L antibody reduced collagen-induced arthritis for an extended period of time compared to PEG sTNFR-1 2.6D alone, as shown in Figure 14.
  • CTLA4-Fc protein and AGP3 Pb (an AGP3 tandem dimer peptide-Fc fusion) used in this study were produced at Amgen. Exemplary AGP3 Pb production is described in Example 4.
  • mice Prior to treatment, mice were pre-screened for protein in the urine with Albustix® reagent strips (Bayer Corp., Elkhart, IN), and those having greater than 100 mg/dl of protein in the urine were included in the experiment. Prior to the injections, murine CTLA4-Fci was suspended in PBS and AGP3 Pb was suspended in PBS.
  • mice Six month old (NZB x NZW) F1 mice were treated intraperitoneally 3 times per week for 12 weeks with 100 ⁇ g (4mg/kg) AGP3 Pb, 50 ⁇ g (2mg/kg) murine CTLA4-Fc, or a combination of 100 ⁇ g (4mg/kg) AGP3 Pb and 50 ⁇ g (2mg/kg) murine CTLA4-Fc.
  • 100 ⁇ g is equivalent to 4 mg of therapeutic per Kg weight of animal (4mg/Kg).
  • 50 ⁇ g is equivalent to 2 mg of therapeutic per Kg weight of animal (2mg/Kg).
  • 100 ⁇ g of human IgGI Fc protein or 100 ⁇ l PBS were administered as controls.
  • mice in the treatment groups were assayed for proteinuria every week using Albustix® reagent strips (Bayer Corp., Elkhart, IN). Each week, the mice were given a score of +1 if their proteinuria became worse (from >100 mg/dl to >300 mg/dl), a score of 0 if their proteinuria stayed the same, and a score of -1 if their proteinuria improved ( ⁇ 100 mg/dl).
  • the mean score for the group of 14 mice is graphed in Figure 16.
  • Figure 16 shows the severity of proteinuria over the course of 41 weeks.
  • the combination treatment of AGP3 Pb and murine CTLA4-Fc reduced the severity of proteinuria compared to administration of AGP3 Pb or murine CTLA4-Fc alone, as shown in Figure 16.
  • CTLA4-Fc protein and AGP3 Pb (an AGP3 tandem dimer peptide-Fc fusion) were produced at Amgen.
  • AGP3 Pb production is described in Example 4.
  • mice Prior to treatment, mice were pre-screened for protein in the urine with Albustix® reagent strips (Bayer Corp., Elkhart, IN), and those having greater than 300 mg/dl of protein in the urine were included in the experiment. Prior to the injections, murine CTLA4-Fc was suspended in PBS and AGP3 Pb was suspended in PBS.
  • mice Six month old (NZB x NZW) F1 mice were treated intraperitoneally 3 times per week for 12 weeks with 100 ⁇ g (4mg/kg) AGP3 Pb, 50 ⁇ g (2mg/kg) murine CTLA4-Fc, or a combination of 100 ⁇ g (4mg/kg) AGP3 Pb and 50 ⁇ g (2mg/kg) murine CTLA4-Fc.
  • 100 ⁇ g is equivalent to 4 mg of therapeutic per Kg weight of animal (4mg/Kg).
  • 50 ⁇ g is equivalent to 2 mg of therapeutic per Kg weight of animal (2mg/Kg).
  • 100 ⁇ g of human IgGI Fc protein or 100 ⁇ l PBS were administered as controls.
  • mice in the treatment groups were assayed for proteinuria every week using Albustix® reagent strips (Bayer Corp., Elkhart, IN). Each week, the mice were given a score of +2 if they died, a score of +1 if their proteinuria worsened (>2000 mg/dl), a score of 0 if their proteinuria stayed the same (300 mg/dl to 2000 mg/dl), a score of -1 if their proteinuria improved ( ⁇ 300 mg/dl to 100 mg/dl), and a score of -2 if their proteinuria improved dramatically ( ⁇ 100 mg/dl).
  • the mean score for the group of 13 mice is graphed in Figure 18.
  • Figure 18 shows the severity of proteinuria over the course of 20 weeks.
  • the combination treatment of AGP3 Pb and murine CTLA4-Fc reduced the severity of proteinuria compared to administration of AGP3 Pb or murine CTLA4-Fc alone, as shown in Figure 18.
  • mice are separated into two groups. In the first group, prior to injection, KIN2 is suspended in A5S and CTLA4-Fc is suspended in PBS. The mice are admistered by injection of 100 ⁇ g of KIN2, 100 ⁇ g CTLA4-FC, or 100 ⁇ g each of KIN2 and CTLA4-Fc at days 0 to +9 post clinical onset of disease. 100 ⁇ g is equivalent to 5 mg of therapeutic per Kg weight of animal (5mg/Kg). Mice are injected in 100 ⁇ l volumes, except in the combination experiment, wherein the mice receive two separate injections of 100 ⁇ l each. Injections of PBS and Fc are used as controls. Mice are monitored once every day and a mean arthritis score is calculated as described in Example 1.
  • mice that score above 1 on the mean arthritis score are treated a second time by injection of 100 ⁇ g of KIN2, 100 ⁇ g CTLA4-Fc, or 100 ⁇ g each of KIN2 and CTLA4-Fc for three additional days. After the second treatment, the three day treatment is repeated whenever a mouse has a mean arthritis score above 1. The treatment is repeated as often as necessary during the course of the experiment.
  • KIN2 is suspended in A5S and CTLA4-Fc is suspended in PBS.
  • the mice are treated by injection of 100 ⁇ g of KIN2, 100 ⁇ g CTLA4-Fc, or 100 ⁇ g each of KIN2 and CTLA4-Fc at days 0 to +2 post clinical onset of disease.
  • 100 ⁇ g is equivalent to 5 mg of therapeutic per Kg weight of animal (5mg/Kg).
  • Mice are injected in 100 ⁇ l volumes, except in the combination experiment, wherein the mice receive two separate injections of 100 ⁇ l each. Injections of PBS and Fc are used as controls. Following treatment mice are evaluated daily and given a mean arthritis score as described in Example 1.
  • mice that score above 1 on the mean arthritis score are treated a second time by injection of a maintenance dose of 100 ⁇ g of KIN2, 100 ⁇ g CTLA4-Fc, or 100 ⁇ g each of KIN2 and CTLA4-Fc for three additional days. After the second treatment, the three day maintenance dose treatment is repeated whenever a mouse has a mean arthritis score above 1. The maintenance dose treatment is repeated as often as necessary during the course of the experiment.
  • mice are separated into two groups.
  • PEG sTNFR-1 2.6D is suspended in PBS and CTLA4-Fc is suspended in PBS.
  • the mice are treated by injection of 100 ⁇ g of PEG sTNFR-1 2.6D, 100 ⁇ g CTLA4-Fc, or 100 ⁇ g each of PEG sTNFR-1 2.6D and CTLA4-Fc at days 0 to +9 post clinical onset of disease.
  • 100 ⁇ g is equivalent to 5 mg of therapeutic per Kg weight of animal (5mg/Kg).
  • Mice are injected in 100 ⁇ l volumes. Injections of PBS and Fc are used as controls. Mice are monitored once every day and a mean arthritis score is calculated as described in Example 1. '
  • mice that score above 1 on the mean arthritis score are treated a second time by injection of a maintenance dose of 100 ⁇ g of PEG sTNFR-1 2.6D, 100 ⁇ g CTLA4-Fc, or 100 ⁇ g each of PEG sTNFR-1 2.6D and CTLA4-Fc for three additional days.
  • the three day maintenance dose treatment is repeated whenever a mouse has a mean arthritis score above 1.
  • the maintenance dose treatment is repeated as often as necessary during the course of the experiment.
  • PEG sTNFR-1 2.6D is suspended in PBS and CTLA4-Fc is suspended in PBS.
  • mice are treated by injection of 100 ⁇ g of PEG sTNFR-1 2.6D, 100 ⁇ g CTLA4-Fc, or 100 ⁇ g each of PEG sTNFR-1 2.6D and CTLA4-Fc at days 0 to +2 post clinical onset of disease.
  • 100 ⁇ g is equivalent to 5 mg of therapeutic per Kg weight of animal (5mg/Kg).
  • Mice are injected in 100 ⁇ l volumes. Injections of PBS and Fc are used as controls. Mice are monitored once every day and a mean arthritis score is calculated as described in Example 1.
  • mice that score above 1 on the mean arthritis score are treated a second time by injection of a maintenance dose of 100 ⁇ g of PEG sTNFR-1 2.6D, 100 ⁇ g CTLA4-Fc, or 100 ⁇ g each of PEG sTNFR-1 2.6D and CTLA4-Fc for three additional days.
  • a maintenance dose 100 ⁇ g of PEG sTNFR-1 2.6D, 100 ⁇ g CTLA4-Fc, or 100 ⁇ g each of PEG sTNFR-1 2.6D and CTLA4-Fc for three additional days.
  • the three day maintenance dose treatment is repeated whenever a mouse has a mean arthritis score above 1.
  • the maintenance dose treatment is repeated as often as necessary during the course of the experiment.
  • PBS and Etanercept is suspended in PBS. Mice are administered by injection of 100 ⁇ g of Etanercept, 100 ⁇ g murine CTLA4-Fc, or 100 ⁇ g each of Etanercept and murine CTLA4-FC at day 0, +1 , +2, +4, +6, +8 and +10 post clinical onset of disease. 100 ⁇ g is equivalent to 5 mg of therapeutic per Kg weight of animal (5mg/Kg). Mice are injected in 100 ⁇ l volumes. PBS and human IgGI Fc (Protein Science, Amgen, Thousand Oaks, CA) are used as controls. Mice are evaluated once a day for a mean arthritic score as described in Example 1.

Abstract

L'invention concerne des méthodes de traitement d'un état inflammatoire ou immunitaire ainsi que des méthodes de traitement d'un état inflammatoire ou immunitaire avec des inhibiteurs d'IL-1 et un inhibiteur de l'activation des lymphocytes B ou T. Des méthodes de traitement d'un état inflammatoire ou immunitaire avec des inhibiteurs de TNF et un inhibiteur de l'activation des lymphocytes B ou T, sont également décrits.
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US7928074B2 (en) 2011-04-19
US20040208874A1 (en) 2004-10-21
WO2004060911A3 (fr) 2005-09-01
MXPA05007019A (es) 2005-08-18
JP2006517191A (ja) 2006-07-20
AU2003299971A1 (en) 2004-07-29
US20080279862A1 (en) 2008-11-13
EP1578782A4 (fr) 2007-09-12
WO2004060911A2 (fr) 2004-07-22
PL378879A1 (pl) 2006-05-29

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